Antisubstances against globulomer aβ, their antigen-binding parts, corresponding hybridomas, nucleic acids, vectors, host cells, ways of production of mentioned antisubstances, compositions containing mentioned antisubstances, application of mentioned antisubstances and ways of application of mentioned antisubstances

FIELD: immunology and bioengineering.

SUBSTANCE: present invention refers to immunology and bioengineering. The variants of an antisubstance that is specific in relation to at least one globulomer Aβ(20-42) have been suggested. Each of the variants is characterized by the fact that it includes VH and VL parts; each of these parts contains three corresponding CDR. The antisubstance antigen-binding section has been revealed. They described: a coding nucleic acid and the vector that contains it, and a host cell that bears the vector that are used for the antisubstance production. The way of antisubstance production with the use of a cell has been discovered. The suggested inventions can find their application in therapy and diagnostics of Alzheimer's disease and other amyloid diseases.

EFFECT: antosubstances that can be used in therapy and diagnostics of Alzheimer's disease and other amyloid diseases.

10 cl, 28 dwg, 9 tbl, 12 ex

 

The invention relates to antibodies against globular Aβ, antigennegative parts, the hybridomas producing these antibodies, nucleic acids encoding these antibodies, vectors containing these nucleic acids, cell host containing the vectors, methods of producing these antibodies, compositions containing these antibodies, therapeutic and diagnostic applications of these antibodies and related methods related to Alzheimer's disease and other amyloidoses.

In 1907 physician Alois Alzheimer first described neurotic diseases signs of a form of dementia, which was later named in his honor (Alzheimer 1907). Alzheimer's disease (AD) is the most frequent cause of dementia among the elderly with an incidence of approximately 10% in the population aged over 65 years. With increasing age the risk of disease also increases. Globally, there are approximately 15 million affected people and expect further increases in life expectancy the number of infected people will increase by about three times over the next decades.

From a molecular point of view of Alzheimer's disease (AD) is characterized by accumulation of abnormally aggregated proteins. In the case of extracellular amyloid plaques these deposits consist mainly of education is of the filaments of the peptide β-amyloid, in the case of intracellular neurofibrillary knots (NFT) from protein tau. The peptide β-amyloid (Aβ) is formed from a protein precursor of β-amyloid peptides by proteolytic cleavage. This splitting occurs due to the cooperative activity of several proteases, designated α-, β - and γ-secretases. Splitting leads to a number of specific fragments of different lengths. Amyloid plaques consist mainly of peptides with a length of 40 or 42 amino acids (Aβ40, Aβ42). The dominant cleavage product is Aβ40; however, Aβ42 has a much stronger toxic effect.

Cerebral deposition of amyloid and cognitive impairment, very similar to the violations observed in Alzheimer's disease, are also signs of down syndrome (trisomy 21), which occurs with a frequency of approximately 1 in 800 births.

Hypothesis the amyloid cascade hardy and Higgins postulates that increased production of Aβ(1-42) will lead to the formation of protofibrils and fibrils, the main components of Aβ plaques, and these fibrils are responsible for the symptoms of Alzheimer's disease. Despite the weak correlation between the severity of dementia and the level of accumulation of Aβ plaques, this hypothesis until recently preferred. Detection of soluble forms of Aβ in the brain in AD, which better correlates with symptoms of AD than ravenblack, led to a revised hypothesis the amyloid cascade.

Active immunization with Aβ peptides reduces the formation of plaque, as well as to the partial decomposition of existing plaques. At the same time it leads to the relief of cognitive impairment on models of transgenic mice APP.

For passive immunization with antibodies directed against Aβ peptides, also found a decrease in the level of Aβ plaques.

The results of the test phase IIa (ELAN Corporation Plc, South San Francisco, CA, USA and Dublin, UK) for active immunization of AN-1792 (peptide Aβ(1-42) in the state of fibrillar aggregation) suggest that immunotherapy against Aβ peptide, was successful. In a subgroup of 30 patients disease progression was significantly reduced in patients with positive titers of antibodies against Aβ as measured by the index MMSE and DAD. However, this study was stopped due to severe side effects in the form of meningoencephalitis (Bennett and Holtzman, 2005, Neurology, 64, 10-12).

Meningoencephalitis is characterized by neuronopathies and infiltration of T-cells in the brain. This is probably due to T-cell immune response induced by the injection of Aβ(1-42) as antigen. This immune response was not expecting after passive immunization. To date there is not yet available clinical data classifies the flax. However, in relation to such a passive method of immunization have expressed concerns regarding the side effect profile due to preclinical studies on very old APP23 mice, which were injected antibody directed against the N-terminal epitope Aβ(1-42) once a week for 5 months. These mice showed an increase in the number and severity of microchromosome compared to control animals treated with saline (Pfeifer et al., 2002, Science, 298, 1379). Also described comparative increase microchromosome very old (> 24 months) mice Tg2576 and PDAPP (Racke et al., 2005, J. Neurosci, 25, 629-636; Wilcock et al. 2004, J. Neuroinflammation, 1(1):24; De Mattos et al., 2004, Neurobiol. Aging 25(S2):577). For both lines of mice injection of antibodies resulted in a significant increase microchromosome. In contrast, the antibody directed against the Central region of the peptide Aβ(1-42), does not induce microchromosome (de Mattos et al., above). The lack of induction of microchromosome was associated with the processing of the antibody, which was not associated with aggregated Aβ peptide in the form of CAA (Racke et al., J. Neurosci, 25, 629-636). However, the exact mechanism leading to microchromosomes in transgenic APP mice is unclear. Perhaps cerebral amyloid angiopathy (CAA) induces or at least increases bleeding in the brain. CAA is present in almost every brain p and Alzheimer's disease, and approximately 20% of cases assessed as "severe CAA". Passive immunization thus should be aimed at excluding microchromosome by selecting antibodies that recognize the Central or C-terminal region of Aβ peptide.

In WO 2004/067561 described stable oligomers Aβ(1-42) (globular Aβ(1-42)) and antibodies specifically directed against globalmenu. Splitting nonspecific proteases showed that globular Aβ can be split, since the hydrophilic N-Terminus protruding from the globular cow patterns (Barghom et al., 2005, J. Neurochem, 95, 834-847). Such truncated from the N-end globular Aβ (Aβ(12-42) and globular Aβ(20-42)) represent the main structural unit of this oligomeric Aβ. They are very vicecommander antigen for active immunization of rabbits and mice, resulting in high titers of antibodies (WO 2004/067561). The alleged pathological role of truncated from the N-Terminus forms of Aβin vivosuggested by several recent reports of their existence in the brain in AD (Sergeant et al., 2003, J. Neurochem, 85, 1581-1591; Thai et al., 1999, J. Neuropathol. Exp Neurol, 58, 210-216). During cleavagein vivomay be involved specific protease, found in the brain, for example, neprilysin (NEP 24.11) or insulindependence enzyme (IDE) (Selkoe, 2001, Neuron, 32, 177-180).

The aim of the present invention was to provide antibodies directed the against globalmenu Aβ, improves cognitive behavior of the patient during immunotherapy, at the same time reacts only with a small part of the total amount of Aβ peptide in the brain. Expect that this will prevent a significant imbalance in cerebral Aβ and cause less side effects. (For example, observed a therapeutically dubious decrease in brain volume in the study of active immunization with Aβ peptides in the state of fibrillar aggregation (test ELAN with AN1792). Moreover, in this test, the observed severe side effects in the form of meningoencephalitis.

The present invention solves this problem by providing globularization antibodies with high affinity for truncated forms of globalmenu Aβ. These antibodies are able to discriminate not only other forms of Aβ peptides, in particular monomers and fibrils, but also the non-truncated forms of globalmenu Aβ.

Thus, the present invention relates to an antibody having binding affinity of for globular Aβ(20-42)greater than the binding affinity of this antibody for globular Aβ(1-42).

In addition, the present invention relates to an antibody having binding affinity of with globularia Aβ(20-42)greater than the binding affinity of this antibody for globular Aβ(12-42).

According to a particular variant of osushestvlyaetsya thus relates to antibodies, possessing binding affinity of for globular Aβ(20-42)greater than the binding affinity of this antibody for globular Aβ(1-42)and globular Aβ(12-42).

The term "Aβ(X-Y)" here refers to the amino acid sequence from the position of the amino acids X to the position of amino acids Y protein β-amyloid person, including both X and Y, in particular to the amino acid sequence from the position of the amino acids X to the position of amino acids Y amino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IAT (consistent with the provisions of amino acids 1-43) or any of its naturally occurring variants, in particular variants with at least one mutation selected from the group consisting of A2T, H6R, D7N, A21G ("Flemish"), E22G ("Arctic"), E22Q ("Dutch"), E22K ("Italian"), D23N ("Iowa"), A42T and A42V, where numbers are shown relative to the start of the Aβ peptide, including both position X and position Y, or sequences containing up to three additional substitutions of amino acids, none of which may prevent the formation of globular, preferably without additional substitutions of amino acids in the portion from amino acid 12 or X, whichever number is higher, to amino acid 42 or Y, depending on what the room below, more preferably, no additional substitutions of amino acids in part from and is inability 20 or X, depending on which number is higher, to amino acid 42 or Y, whichever number is lower, and most preferably, no more replacement amino acids in the portion from amino acid 20 or X, whichever number is higher, to amino acid 40 or Y, depending on what the room below, where "additional" replacement of amino acids there is any deviation from the canonical sequence not found in nature.

More specifically, the term "Aβ(1-42)" here refers to the amino acid sequence from the position of amino acids 1 to the provisions of 42 amino acids in the protein β-amyloid, including both 1 and 42, in particular to the amino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA or any of its naturally occurring variants, in particular variants with at least one mutation selected from the group consisting of A2T, H6R, D7N, A21G ("Flemish"), E22G ("Arctic"), E22Q ("Dutch"), E22K ("Italian"), D23N ("Iowa"), A42T and A42V, where numbers are shown relative to the start of the Aβ peptide, including both 1 and 42, or a sequence that contains up to three additional substitutions of amino acids, none of which may prevent the formation of globular, preferably without additional substitutions of amino acids in the portion from amino acid 20 to amino acid 42. Similarly, those who min "Aβ(1-40)" here refers to the amino acid sequence from the position of amino acids 1 to the provisions of 40 amino acids of the protein β-amyloid person, including both 1 and 40, in particular to the amino acid sequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV or any of its naturally occurring variants, in particular variants with at least one mutation selected from the group consisting of A2T, H6R, D7N, A21G ("Flemish"), E22G ("Arctic"), E22Q ("Dutch"), E22K ("Italian"), D23N ("Iowa"), where numbers are shown relative to the start of the Aβ peptide, including both 1 and 40, or a sequence that contains up to three additional substitutions of amino acids, none of which may prevent the formation of globular, preferably, no more replacement amino acids in the portion from amino acid 20 to amino acids 40.

More specifically, the term "Aβ(12-42)" here refers to the amino acid sequence from position 12 amino acids to the provisions of 42 amino acids of the protein β-amyloid person, including both 12 and 42, in particular to the amino acid sequence VHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA or any of its naturally occurring variants, in particular variants with at least one mutation selected from the group consisting of A21G ("Flemish"), E22G ("Arctic"), E22Q ("Dutch"), E22K ("Italian"), D23N ("Iowa"), A42T and A42V, where numbers are shown relative to the start of the Aβ peptide, including both 12 and 42, or a sequence containing up to three additional C is Myung-amino acids, none of them can prevent the formation of globular, preferably, no more replacement amino acids in the portion from amino acid 20 to amino acid 42.

More specifically, the term "Aβ(20-42)" here refers to the amino acid sequence from the position of amino acids 20 to the provisions of 42 amino acids of the protein β-amyloid person, including both 20 and 42, in particular to the amino acid sequence F AEDVGSNKGA IIGLMVGGVV IA or any of its naturally occurring variants, in particular variants with at least one mutation selected from the group consisting of A21G ("Flemish"), E22G ("Arctic"), E22Q ("Dutch"), E22K ("Italian"), D23N ("Iowa"), A42T and A42V, where numbers are shown relative to the start of the Aβ peptide, including both 20 and 42, or a sequence that contains up to three additional substitutions of amino acids, none of which may prevent the formation of globular, preferably without any additional substitutions of amino acids.

The term "globular Aβ(X-Y)" (globular oligomer Aβ(X-Y)) here refers to a soluble, globular, non-covalent Association of the peptides Aβ(X-Y), as defined above, with homogeneity and distinct physical characteristics. According to one aspect of globular Aβ(X-Y) are stable, defibrillate, oligomeric, ansambl the peptides Aβ(X-Y), which can be obtained by incubation with anionic detergents. Unlike monomers and fibrils, these Globaloney are characterized by a certain number of subunits in the ensemble (for example, forms of early ensembles, n=4-6, "oligomers A"form of late ensembles, n=12-14, "oligomers B", as described in WO2004/067561). Globaloney have 3-dimensional structure of globular type ("molten globule", see Barghorn et al., 2005, J. Neurochem, 95, 834-847). They can further be characterized by one or more of the following properties:

- an opportunity removal of N-terminal amino acids X-23 nonspecific proteases (such as thermolysin or endoprotease GluC) to obtain the truncated forms of globalmenu;

- unavailability of C-terminal amino acids 24-Y for non-specific proteases and antibodies;

- truncated forms of these globalmenu support 3-dimensional crustal structure of these globalmenu with the best availability crustal epitope Aβ(20-Y) in its globalmenu conformation.

According to the invention and, in particular, for the purposes of assessing affinely binding antibodies of the present invention, the term "globular Aβ(X-Y)" here refers in particular to the product, which can be obtained by the method as described in WO 2004/067561, the contents of which are hereby incorporated by reference.

This method includes maximizing natural, recombinant the th or synthetic peptide Aβ(X-Y) or its derivative; exposure to at least partially deployed peptide Aβ(X-Y) or its derivative to the influence of detergent, reducing the action of the detergent and continued incubation.

For the purposes of maximizing peptide can prevent the effect on protein destructive hydrogen bond funds, such as hexafluoroisopropanol (HFIP). The exposure time to a few minutes, for example about 10-60 minutes, enough, when the exposure temperature is from about 20 to 50°C and in particular, about 35-40°C., followed by dissolving the precipitate, evaporated to dryness, preferably in concentrated form, in suitable organic solvents that can mix with water buffers, such as dimethylsulfoxide (DMSO), get the suspension is at least partially deployed peptide or its derivative, which can then be used. If necessary, the original suspension can be stored at a low temperature, for example, approximately at -20°C over a time period.

Alternatively, the peptide or its derivative can be placed in a weak acid, preferably aqueous, solution of, for example, in an aqueous solution of approximately 10 mm HCl. After the incubation time, usually several minutes, insoluble components are removed by centrifugation. Come a few minutes at 10,000 g. the t stage of the method is preferably carried out at room temperature, i.e. a temperature in the range from 20 to 30°C. the Supernatant obtained after centrifugation contains the peptide Aβ(X-Y) or its derivative, and can be stored at a low temperature, for example, approximately at -20°C over a time period.

Following the impact of detergent relates to the oligomerization of the peptide or its derivative to obtain an intermediate type oligomers (indicated in WO 2004/067561 as oligomers (A). For this purpose, the detergent allows to act on the at least partially unfolded peptide or its derivative to obtain a sufficient number of intermediate oligomer.

Preference to have the use of ionic detergents, in particular anionic detergents.

According to a particular variant of the implementation use a detergent of the formula (I):

R-X

in which the radical R represents an unbranched or branched alkyl having from 6 to 20 and preferably 10 to 14 carbon atoms, or an unbranched or branched alkenyl having from 6 to 20 and preferably 10 to 14 carbon atoms, the radical X represents an acidic group or its salt, with X preferably selected from-COO-M+, -SO3-M+and especially OSO3-M+and M+represents a cation, hydrogen or an inorganic or organic to the tion, preferably selected from the cations of the alkali metal and alkaline earth metal and ammonium cations.

Predominant are the detergents of the formula (I)in which R is an unbranched alkyl, in which, in particular, need to be mentioned alkyl radicals. Particular preference is given to sodium dodecyl sulfate (SDS). Preferably you can also use lauric acid and oleic acid. Sodium salt detergent laurylsarcosine (also known as sarkosyl NL-30 or Gardol®) is particularly preferred.

The exposure time of detergent depends, in particular, on whether - and if it is, to what extent, the peptide or its derivative, subjected to oligomerization deployed. If according to the stage of deployment of the peptide or its derivative pretreated destroying the hydrogen bonds means, i.e. in particular, hexafluoroisopropanol, exposure time in the range of a few hours, mostly from approximately 1 to 20 and in particular from about 2 to 10 hours, enough with the exposure temperature of approximately 20-50°C and, in particular, approximately 35-40°C. If the starting point is nesvorny or basically not folded peptide or its derivative, respectively, a longer exposure time is CE is EcoObraz. If the peptide or its derivative pre-treated, for example, according to the method mentioned above, alternatively, the processing HFIP, or the specified peptide or its derivative directly subjected to oligomerization, enough time exposure in the range of from about 5 to 30 hours and in particular from about 10 to 20 hours, when the exposure temperature is approximately 20-50°C and, in particular, approximately 35-40°C. After incubation, insoluble components are preferably removed by centrifugation. A few minutes at 10000 g are appropriate.

Select the concentration of detergent used depends on the detergent. If used SDS, it is proved that the concentration in the range of from 0.01 to 1% by weight, preferably from 0.05 to 0.5% by weight, for example about 0.2% by weight, is suitable. If used lauric acid or oleic acid, are suitable are slightly higher concentration, for example, in the range from 0.05 to 2% by weight, preferably from 0.1 to 0.5% by weight, for example, about 0.5 mass%.

The action of the detergent must occur when the salt concentration approximately in the physiological range. Thus, are appropriate NaCl concentration, in particular in the range from 50 to 500 mm, preferably from 100 is about 200 mm and, in particular, approximately 140 mm.

Subsequent reduction of the action of the detergent and continued incubation refers to further oligomerization to obtain globular Aβ(X-Y) according to the invention (WO 2004/067561 labeled oligomers (B). Because the composition obtained in the previous stages, typically contains a detergent and a salt concentration in the physiological range, it is reasonable to reduce the effect of the detergent and, preferably, also the salt concentration. This can be done by reducing the concentration of detergent and salt, such as breeding, it is advisable water or buffer with a lower concentration of salts, such as Tris-HCl, pH 7.3. It is proved that the fit factors of cultivation, ranging from approximately 2 to 10, mainly in the range from approximately 3 to 8 and, in particular, approximately 4. To reduce the action of detergent can also be achieved by adding substances that can neutralize the specified action of the detergent. Examples of substances include substances capable of forming complexes with a detergent, such as substances capable of stabilizing cells during cleaning methods and selection, for example, a particular block EO/PO copolymers, in particular block copolymers under the trade name Pluronc® F 68. Similarly, you can use alkoxysilane and the particular, ethoxylated ALKYLPHENOLS, such as ethoxylated t-ops series Triton® X, in particular Triton® X100, 3-(3-chloromethylketone)-1-propanesulfonate (CHAPS®) or alkoxysilane and, in particular, ethoxylated fatty esters sorbitan, such as esters of a series of Tween®, in particular Tween® 20, in the concentration ranges in area or greater than a certain critical concentration of micelles.

Then the solution is incubated to obtain a sufficient number of globular Aβ(X-Y) according to the invention. Time steps in the range of a few hours, preferably in the range of from about 10 to 30 hours, and in particular in the range from approximately 15 to 25 hours, enough, when the temperature of action is approximately 20-50°C and, in particular, approximately 35-40°C. the Solution can then be concentrated and possible precipitation can be removed by centrifugation. It is proved that here are suitable for a few minutes at 10,000 g. The supernatant obtained after centrifugation contains globular Aβ(X-Y) according to the invention.

Globular Aβ(X-Y) according to the invention it is possible to completely isolate in pure form in a known manner, for example, ultrafiltration, dialysis, precipitation or centrifugation.

In addition, it is preferred if the electrophoretic separation of globalmenu Aβ(X-Y) in dent the generating conditions, for example, SDS-PAGE, get a dual band (for example, with an apparent molecular mass of 38 / 48 kDa for Aβ(1-42)), and particularly preferably, if the processing glutardialdehyde of globalmenu before separating the two lanes merge into one. Also is preferred if exclusion chromatography of globalmenu receive a separate peak (for example, corresponding to molecular mass of approximately 100 kDa for globular Aβ(1-42) or approximately 60 kDa cross-linked glutardialdehyde of globular Aβ(1-42)), respectively.

Since the peptide Aβ(1-42)peptide Aβ(12-42) and peptide Aβ(20-42), these methods, in particular, are suitable for obtaining globalmenu Aβ(1-42), globularia Aβ(12-42) and globularia Aβ(20-42).

In a specific embodiment, the invention globular Aβ(X-Y), where X is selected from the group consisting of the numbers 2.. 24 and Y is as defined above, are those which can be obtained by truncation globalmenu Aβ(1-Y) to shorter forms, where X is selected from the group consisting of the numbers 2.. 24, where X preferably represents a 20 or 12, and Y is as defined above, which can be obtained by processing the respective proteases. For example, globular Aβ(20-42) can be obtained by subjecting globular Aβ(1-42) proteolysis by thermolysin, and globular Aβ(12-42) you shall receive, exposing globular Aβ(1-42) proteolysis by endoprotease GluC. Upon reaching the desired degree of proteolysis protease inactivate well-known manner. The resulting globular then you can select according to the methods already described here and, if necessary, through additional processing through additional stages of exposure and cleaning. A detailed description of these methods are described in WO 2004/067561, the contents of which are hereby incorporated by reference.

For the purposes of the present invention globular Aβ(1-42), in particular, is globular Aβ(1-42), as described in example 1a here; globular Aβ(20-42), in particular, is globular Aβ(20-42), as described in examples 1c here, and globular Aβ(12-42), in particular, is globular Aβ(12-42), as described in examples 1d here.

Preferably, for globularia show affinity for neuronal cells. Preferably, for globularia also show neyromoduliruyuschim effects.

According to another aspect of the invention, globular consists of 11-16 and, most preferably, from 12-14 peptides Aβ(X-Y).

According to another aspect of the invention, the term "globular Aβ(X-Y)" here refers to globular, consisting mainly of subunits Aβ(X-Y), which is preferred, if on average at least 11 of the 12 subunits of the principles of delegat type Aβ(X-Y), more preferably, if less than 10% of globalmenu contain any unrelated to Aβ(X-Y) peptides and, most preferably, if the content is not related to Aβ(X-Y) peptides below the threshold of detection.

More specifically, the term "globular Aβ(1-42)" here refers to globular, consisting mainly of units of Aβ(1-42), as defined above; the term "globular Aβ(12-42)" here refers to globular, consisting mainly of units of Aβ(12-42), as defined above; and the term "globular Aβ(20-42)" here refers to globular, consisting mainly of units of Aβ(20-42), as defined above.

The term "cross-stitched globular Aβ(X-Y)" here refers to a molecule that can be obtained from globular Aβ(X-Y), as described above, by means of cross-stitching, preferably, the chemical cross-linkage, more preferably, cross-stitching aldehyde, most preferably, cross-stitching glutardialdehyde components of globular units. In another aspect of the invention cross-stitched globular basically is globular where units are at least partially connected by covalent bonds, and not held together through non-covalent interactions. For the purposes of the present invention cross-stitched globules which measures Aβ(1-42) is a in particular, cross-linked oligomer Aβ(1-42), as described in example 1b here.

The term "derived globular Aβ(X-Y)" here refers in particular to globalmenu, which is labeled by covalent binding with a group that facilitates detection, preferably a fluorophore, for example, fluoresceinisothiocyanate, phycoerythrin, fluorescent protein of Aequorea victoria fluorescent protein Dictyosoma or any combination thereof, or fluorescently active derivative; a chromophore; chemiluminometer, for example luciferase, preferably a luciferase Photinus pyralis, Vibrio fischeri luciferase, or any combination thereof or chemiluminescence active derivative; enzymatically active group, for example, peroxidase such as horseradish peroxidase, or any of their enzymatically active derivative; electronmobility group, for example, a group containing a heavy metal, for example, a group containing gold; a hapten, for example, obtained from phenol by hapten; the structure is strong antigen, for example a peptide sequence, as predicted, which is antigenic, for example, which is antigenic, as predicted by the algorithm Kolaskar and Tongaonkar; the aptamer for the other molecules; chelat forming group, such as hexaglycine; natural or derived from natural protein structure mediating complement the global protein-protein interaction, for example, a member of a pair of fos/jun; a magnetic group, for example a ferromagnetic group; or a radioactive group, for example group containing1H,14C,32P,35S or125I, or any combination; or to globalmenu, marked by covalent or non-covalent high-affinity interaction, preferably covalently associated with a group that facilitates inactivation, sequestration, degradation and/or precipitation, preferably marked by the group, stimulating the degradation ofin vivomore preferably, the ubiquitin, which is particularly preferred is that this labeled oligomer was collected byin vivo; or globular, modified by any combination of the above. Such groups for marking and labeling, and how to attach them to proteins known in this field. Labelling and/or marking can be performed before, during or after globularization. In another aspect of the invention derived globularia is a molecule that can be obtained from globularia through reaction labelling and/or marking.

Accordingly, the term "derivative monomer Aβ(X-Y)" here refers in particular to the Aβ monomer, which is labeled or marked as described for globularia.

It is advantageous if the antibody truly is obreteniyu links globular Aβ(20-42) with K Din the range from 1×10-6M to 1×10-12M. Preferably, the antibody binds to globular Aβ(20-42) with high affinity, for example with KD1×10-7M or greater affinity, e.g. with KD3×10-8M or greater affinity, with KD1×10-8M or greater affinity, e.g. with a KD of 3×10-9M or greater affinity, with KD1×10-9M or greater affinity, e.g. with KD3×10-10M or greater affinity, with KD1×10-10M or greater affinity, e.g. with KD3×10-11M or greater affinity, or with KD1×10-11M or greater affinity.

The term "greater affinity" here refers to the degree of interaction, where the equilibrium between unbound antibody and unbound globularia, on the one hand, and a complex of the antibody-globular, on the other, more shifted towards the complex of antibody-globular. Similarly, the term "lower affinity" here refers to the degree of interaction, where the equilibrium between unbound antibody and unbound globularia, on the one hand, and a complex of the antibody-globular, on the other, more shifted towards the unbound antibody and unbound globularia. The term "greater affinity" is synonymous with the term "higher affinity", and the term "lower affinity" is a synonym Ter is in the "low affinity".

According to a particular variant implementation of the invention relates to antibody binding globular Aβ(20-42) with KDin the range from 1×10-6M to 1×10-12M, globular Aβ(1-42) with KD10-12M or smaller affinity, where the affinity of binding to globularia Aβ(20-42) greater than the binding affinity of with globularia Aβ(1-42).

It is preferable that the binding affinity of antibodies of the present invention with globularia Aβ(20-42) at least 2 times, such as at least 3 times or at least 5 times, preferably at least 10 times, for example at least 20 times, at least 30 times or at least 50 times, more preferably at least 100 times, for example, at least 200 times, at least 300 times or at least 500 times, and even more preferably at least 1000 times, for example at least 2000 times, at least 3000 times or at least 5000 times, even more preferably at least 10,000 times, for example, at least 20000 times, at least 30000 or at least 50000 times, and most preferably at least 100000 times greater than the binding affinity of antibodies with globularia Aβ(1-42).

According to a particular variant implementation of the invention relates to antibody binding globular Aβ(12-42) with K D10-12M or smaller affinity, where the affinity of binding to globularia Aβ(20-42) greater than the binding affinity of with globularia Aβ(12-42).

It is preferred that the binding affinity of antibodies of the present invention with globularia Aβ(20-42) at least 2 times, such as at least 3 times or at least 5 times, preferably at least 10 times, for example at least 20 times, at least 30 times or at least 50 times, more preferably at least 100 times, for example, at least 200 times, at least 300 times or at least 500 times, and even more preferably at least 1000 times, for example at least 2000 times, at least 3000 times or at least 5000 times, even more preferably at least 10,000 times, for example, at least 20000 times, at least 30000 or at least 50000 times, and most preferably at least 100000 times greater than the binding affinity of antibodies with globularia Aβ(12-42).

Preferably, antibodies of the present invention bind at least one globular Aβ, as defined above, and have relatively lower affinity for at least one non-globular form of Aβ.

Antibodies of the present invention, having relatively IU the greater affinity for at least one non-globular forms of Aβ, than at least one of globular Aβ include antibodies having binding affinity of with globularia Aβ(20-42)greater than the affinity for the monomer Aβ(1-42). In addition, it is preferable that, alternatively or additionally, the binding affinity of antibodies with globularia Aβ(20-42) was greater than the affinity for the monomer Aβ(1-40).

In a preferred embodiment according to the invention the affinity of antibodies for globular Aβ(20-42) exceeds its affinity as a monomer Aβ(1-40)and Aβ monomer(1-42).

The term "monomer Aβ(X-Y)" here refers to the selected form of the peptide Aβ(X-Y), preferably in the form of peptide Aβ(X-Y), which do not interact mainly non-covalent interactions with other Aβ peptides. In practice, the monomer Aβ(X-Y) are usually obtained in the form of an aqueous solution. In a particularly preferred embodiment according to the invention an aqueous solution of the monomer contains 0.05%-0,2%, more preferably, about 0.1% NH4OH. In another particularly preferred variant of the invention, the aqueous monomer solution contains 0.05%-0,2%, more preferably, about 0.1% NaOH. When using (for example, to determine affinely binding antibodies of the present invention), it may be advisable to dilute this solution appropriately. In addition, Oba is but it is advisable to use this solution for 2 hours, in particular, within 1 hour, and especially within 30 minutes after its preparation.

More specifically, the term "monomer Aβ(1-40)" here refers to the preparation of the monomer Aβ(1-40), as described in example 2 here, the term "monomer Aβ(1-42)" here refers to the drug Aβ(1-42), as described in example 2 here.

It is recommended that the antibody of the present invention were connected by one or, more preferably, both of the monomer with low affinity, most preferably, with KD1×10-8M or smaller affinity, for example, with KD3×10-8M or smaller affinity, with KD1×10-7M or smaller affinity, for example, with KD3×1-7M or smaller affinity, or with KD1×10-6M or smaller affinity, for example, with KD3×10-5M or smaller affinity, or with KD1×10-5M or smaller affinity.

Especially, it is preferable that the binding affinity of antibodies of the present invention with globularia Aβ(20-42) at least 2 times, such as at least 3 times or at least 5 times, preferably at least 10 times, for example at least 20 times, at least 30 times or at least 50 times, more preferably at least 100 times, for example at least 200 times, at least 300 times or at least 500 times, and Yes the e is more preferably at least 1000 times, for example at least 2000 times, at least 3000 times or at least 5000 times, even more preferably at least 10,000 times, for example, at least 20000 times, at least 30000 or at least 50000 times, and most preferably at least 100000 times greater than the binding affinity of antibodies to one or, more preferably, both monomers.

In addition, antibodies of the present invention, having a relatively lower affinity for at least one non-globular forms of Aβ than at least one of globular Aβ additionally include antibodies having binding affinity of with globularia Aβ(20-42)greater than the affinity of binding to fibrils of Aβ(1-42). In addition, it is preferable that, alternatively or additionally, the binding affinity of antibodies with globularia Aβ(20-42) was greater than the affinity of binding to fibrils of Aβ(1-40).

The term "fiber" here refers to the molecular structure containing ensembles ecovalence related individual peptides Aβ(X-Y), which shows a fibrillar structure in the electron microscope, the binding of Congo red, and then birefringence under polarized light and which by the nature of rentgenodiffraction is a crosslinked β-is the structure.

In another aspect of the invention, the fiber is a molecular structure that can be obtained by a method that includes self-induced polymer aggregation of the corresponding Aβ peptide in the absence of detergents, for example, 0.1 M HCl, resulting in the formation of aggregates of more than 24, preferably more than 100 units. These methods are well known in this field. It is advisable to use the fibrils of Aβ(X-Y) in the form of an aqueous solution. In a particularly preferred variant of the invention, the aqueous solution of fibrils obtained by dissolution of Aβ peptide in 0.1% NH4OH, dilution 1:4 in 20 mm NaH2PO4, 140 mm NaCl, pH 7,4, then bringing the pH to 7.4, the incubation solution at 37°C for 20 h, followed by centrifugation at 10000 g for 10 min and resuspending 20 mm NaH2PO4, 140 mm NaCl, pH 7,4.

The term "fiber Aβ(X-Y)" here refers to a fiber consisting mainly of subunits Aβ(X-Y), where it is preferable, if on average at least 90% of the subunits are subunits of type Aβ(X-Y), more preferably, if at least 98% of the subunits are subunits of type Aβ(X-Y), and most preferably, if the content is not related to Aβ(X-Y) peptides below the threshold of detection.

More specifically, the term "fiber Aβ(1-42)" here refers the I to the drug fibrils Aβ(1-42), as described in example 3 here.

It is recommended that the antibody of the present invention were connected by one or, more preferably, both fibrils with low affinity, most preferably KD1×10-8M or smaller affinity, for example with KD3×10-8M or smaller affinity, with KD1×10-7M or smaller affinity, for example with KD3×10-7M or smaller affinity, or with KD1×10-6M or smaller affinity, for example, with KD3×10-5M or smaller affinity, or with KD1×10-5M or smaller affinity.

Especially, it is preferable that the binding affinity of antibodies of the present invention with globularia Aβ(20-42) at least 2 times, such as at least 3 times or at least 5 times, preferably at least 10 times, for example at least 20 times, at least 30 times or at least 50 times, more preferably at least 100 times, for example, at least 200 times, at least 300 times or at least 500 times, and even more preferably at least 1000 times, for example at least 2000 times, at least 3000 times or at least 5000 times, even more preferably at least 10,000 times, for example, at least 20000 times, at least 30000 or less the th least 50,000 times, and most preferably at least 100000 times greater than the binding affinity of antibodies with one or, more preferably, both fibrils.

According to one of specific embodiments the invention relates to antibodies having binding affinity of with globularia Aβ(20-42)greater than its binding affinity of both fibrils Aβ(1-40)and Aβ fibrils(1-42).

According to a particularly preferred variant implementation of the present invention relates to antibodies with relatively lower affinity for Monomeric and fibrillar forms of Aβ than at least one of globular Aβ, in particular globular Aβ(20-42). These antibodies then here indicate how specific globular antibodies.

In addition, antibodies of the present invention include antibodies having binding affinity of with globularia Aβ(20-42)greater than the binding affinity of cross-stitched globular Aβ(1-42), in particular for cross-linked glutardialdehyde of globular Aβ(1-42), such as described in example 1b here.

In a particularly preferred embodiment according to the invention the binding affinity of antibodies with globularia Aβ(20-42) at least 2 times, such as at least 3 times or at least 5 times, predpochtitel is but at least 10 times, for example at least 20 times, at least 30 times or at least 50 times, more preferably at least 100 times, for example at least 200 times, at least 300 times or at least 500 times, and even more preferably at least 1000 times, for example at least 2000 times, at least 3000 times or at least 5000 times, even more preferably at least 10,000 times, for example, at least 20000 times, at least 30000 or at least 50000 times, and most preferably at least 100000 times greater than the binding affinity of antibodies with cross-stitched globularia Aβ(1-42).

Antibodies of the present invention are preferably selected, in particular monoclonal and, more specifically, recombinant.

The present invention also relates to a monoclonal antibody (5F7), which can be obtained from hybridoma outlined in the American type culture collection number Deposit PTA-7241.

The present invention also relates to a monoclonal antibody (10F11), which can be obtained from hybridoma outlined in the American type culture collection number Deposit PTA-7239.

The present invention also relates to a monoclonal antibody (7C6), which can be obtained from hybridoma about the landscapes in the American type culture collection number Deposit PTA-7240.

The present invention also relates to a monoclonal antibody (4B7), which can be obtained from hybridoma outlined in the American type culture collection number Deposit PTA-7242.

The present invention also relates to a monoclonal antibody (6A2), which can be obtained from hybridoma outlined in the American type culture collection number Deposit PTA-7409.

The present invention also relates to a monoclonal antibody (2F2), which can be obtained from hybridoma outlined in the American type culture collection number Deposit PTA-7408.

The present invention also relates to a monoclonal antibody (4D10), which can be obtained from hybridoma outlined in the American type culture collection number Deposit PTA-7405.

The present invention also relates to a monoclonal antibody (7E5), which can be obtained from hybridoma outlined in the American type culture collection number Deposit PTA-7809.

The present invention also relates to a monoclonal antibody (10C1), which can be obtained from hybridoma outlined in the American type culture collection number Deposit PTA-7810.

The present invention also relates to a monoclonal antibody (3B10) which can be obtained from hybridoma identified in the AMA is the ikan type culture collection number Deposit PTA-7851.

These antibodies of the present invention, 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10, characterized as having a binding affinity of with globularia Aβ(20-42)greater than the binding affinity of this antibody for globular Aβ(1-42).

The present invention also relates to antibodies having the same profile link, as any of these monoclonal antibodies 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10. Antibodies having the same profile link, as any of these monoclonal antibodies, should not be construed as limited to antibodies having binding affinity of with globularia Aβ(20-42)greater than the binding affinity of this antibody for globular Aβ(1-42).

Antibodies having the same profile link, as any of these monoclonal antibodies 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10 include antibodies that bind the same epitope as monoclonal antibodies 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10.

All monoclonal antibodies from the group consisting of 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10, bind the epitope is contained within the range of the sequence 20 and 42 of Aβ, in particular within the range of the sequence 20-30 Aβ. Without communication with theory, believe that the epitope is a structural, not a linear epitope between subunits in the region of amino acids 20 and 42,in particular in the field of amino acids 20 and 30.

The present invention also relates to antibodies capable of competing with at least one, preferably all, of the antibodies selected from the group consisting of 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10.

The term "competing antibody" here refers to any number of antibodies directed against the same molecular or ecovalence related supramolecular entity, preferably, of the same molecule, where at least one is able to specifically reduce the measured binding of another, preferably by steric interference to access another antibody to the epitope to which it is aimed, or guidance, and/or stabilize the conformation of the group target, which reduces the affinity of the target for the other antibodies, more preferably, by direct block access of the second antibody to its epitope of the target by binding epitope in enough close proximity to the epitope for the first, overlapping with the epitope for the first or the same epitope for the first, most preferably, overlapping, or identical to, especially, are identical. Here they say that the two epitopes are "overlapping"if they share part of their chemical structure, preferably their amino acid sequence, and are "identical", e is whether their chemical structure, preferably the amino acid sequences are identical.

Thus, the present invention also relates to antibodies, epitopes of the target which are overlapping, preferably identical epitope-targeted by at least one antibody selected from the group consisting of 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10.

Antibodies with the same profile link profile of any of these monoclonal antibodies 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10, thereby further include antibodies that contains at least part of antigennegative part of any of these monoclonal antibodies 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10. Preferably, the portion comprises at least one complementarity determining region (CDR) of any of the above monoclonal antibodies 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10.

Thus, according to further specific variant implementation, the present invention relates to antibodies containing the amino acid sequence of CDR3 of the heavy chain and/or amino acid sequence of CDR3 light chain monoclonal antibodies 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 or 3B10. Specific examples of such antibodies include antibodies that also contain the amino acid sequence of CDR2 of the heavy chain is/or amino acid sequence of light chain CDR2 of monoclonal antibodies 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 or 3B10, respectively. More specifically, such antibodies include antibodies that also contain the amino acid sequence of CDR1 of the heavy chain and/or the amino acid sequence of the light chain CDR1 of monoclonal antibodies 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 or 3B10, respectively.

Thus, in one aspect the present invention relates to an antibody containing heavy chain, where domain CDR3, CDR2, and/or contains CDR1 amino acid sequence of CDR3, CDR2, and/or CDR1 of the heavy chain of the monoclonal antibody 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 or 3B10.

Thus, in this aspect the present invention relates to an antibody containing light chain, where the domain CDR3, CDR2, and/or contains CDR1 amino acid sequence of CDR3, CDR2 and/or light chain CDR1, respectively, monoclonal antibodies 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 or 3B10.

Preferably, the antibody contains at least one CDR that contains the amino acid sequence selected from the group consisting of: amino acid residues 31-35 of SEQ ID NO:3, amino acid residues 50-66 of SEQ ID NO:3, amino acid residues 99-109 of SEQ ID NO:3, amino acid residues 24-39 of SEQ ID NO:4, amino acid residues 55-61 of SEQ ID NO:4, amino acid residues 94-102 of SEQ ID NO:4, amino acid residues 31-35 of SEQ ID NO:7, amino acid residues 50-66 of SEQ ID NO7, amino acid residues 97-109 of SEQ ID NO:7, amino acid residues 24-39 of SEQ ID NO:8, amino acid residues 55-61 of SEQ ID NO:8, amino acid residues 94-102 of SEQ ID NO:8, amino acid residues 31-35 of SEQ ID NO:11, amino acid residues 50-65 of SEQ ID NO:11, amino acid residues 98-107 of SEQ ID NO:11, amino acid residues 24-39 of SEQ ID NO:12, amino acid residues 55-61 of SEQ ID NO:12, amino acid residues 94-102 of SEQ ID NO: 12, amino acid residues 31-35 of SEQ ID NO: 15, amino acid residues 50-66 of SEQ ID NO: 15, amino acid residues 99-107 of SEQ ID NO: 15, amino acid residues 24-40 of SEQ ID NO: 16, amino acid residues 56-62 of SEQ ID NO: 16, amino acid residues 95-103 of SEQ ID NO: 16, amino acid residues 31-35 of SEQ ID NO: 19, amino acid residues 50-66 of SEQ ID NO: 19, amino acid residues 99-109 of SEQ ID NO: 19, amino acid residues 24-39 of SEQ ID NO:20, amino acid residues 55-61 of SEQ ID NO:20, amino acid residues 94-102 of SEQ ID NO:20, amino acid residues 31-35 of SEQ ID NO:23, amino acid residues 50-66 of SEQ ID NO:23, amino acid residues 99-109 of SEQ ID NO:23, amino acid residues 24-39 of SEQ ID NO:24, amino acid residues 55-61 of SEQ ID NO:24, amino acid residues 94-102 of SEQ ID NO:24, amino acid residues 31-35 of SEQ ID NO:27, amino acid residues 50-65 of SEQ ID NO:27, amino acid residues 98-101 of SEQ ID NO:27, amino acid residues 24-39 of SEQ ID NO:28, amino acid residues 55-61 of SEQ ID NO:28, amino acid mod the s 94-102 of SEQ ID NO:28, amino acid residues 31-35 of SEQ ID NO:31, amino acid residues 50-66 of SEQ ID NO:31, and amino acid residues 99-107 of SEQ ID NO:31, and amino acid residues 24-40 of SEQ ID NO:32, amino acid residues 56-62 of SEQ ID NO:32, amino acid residues 95-103 of SEQ ID NO:32, amino acid residues 31-35 of SEQ ID NO:35, amino acid residues 50-66 of SEQ ID NO:35, amino acid residues 99-107 of SEQ ID NO:35, amino acid residues 24-40 of SEQ ID NO:36, amino acid residues 56-62 of SEQ ID NO:36, amino acid residues 95-103 of SEQ ID NO:36, amino acid residues 31-35 of SEQ ID NO:38, amino acid residues 50-66 of SEQ ID NO:38, amino acid residues 98-109 of SEQ ID NO:38.

In a preferred embodiment, the antibody comprises at least 3 CDRs selected from the group consisting of the sequences described above. More preferably, 3 CDR selected from CDR sets variable domains selected from the group consisting of:

VH 5F7 set CDR
VH 5F7 CDR-H1TFYIH:
Residues 31-35 of SEQ ID NO:3
VH 5F7 CDR-H2MIGPGSGNTYYNEMFKD:
Residues 50-66 of SEQ ID NO:3
VH 5F7 CDR-H3AKSARAAWFAY:
Residues 99-109 of SEQ ID NO:3
VL 5F7 set CDR
VL 5F7 CDR-L1RSSQSVVQSNGNTYLE:
Residues 24-39 of SEQ ID NO:4
VL 5F7 CDR-L2KVSNRFS:
Residues 55-61 of SEQ ID NO:4
VL 5F7 CDR-L3FQGSHVPPT:
Residues 94-102 of SEQ ID NO:4

VH 10F11 set CDR
VH 10F11 CDR-H1SYVMH:
Residues 31-35 of SEQ ID NO:7
VH 10F11 CDR-H2YIYPYNDGTKYNEKFKG:
Residues 50-66 of SEQ ID NO:7
VH 10F11 CDR-H3TVEGATWDGYFDV:
Residues 97-109 of SEQ ID NO:7
VL 10F11 set CDR
VL 10F11 CDR-L1KSSQSLLYSKGKTYLN:
Residues 24-39 of SEQ ID NO:8
VL 10F11 CDR-L2LVSKLDS:
Residues 55-61 of SEQ ID NO:8
VL 10F11 CDR-L3VQGTHFPHT:
Residues 94-102 of SEQ ID NO:8
VH 7C6 set CDR
VH 7C6 CDR-H1SYAMS:
Residues 31-35 of SEQ ID NO:11
VH 7C6 CDR-H2 SIHNRGTIFYLDSVKG:
Residues 50-65 of SEQ ID NO:11
VH 7C6 CDR-H3GRSNSYAMDY:
Residues 99-107 of SEQ ID NO:11
VL 7C6 set CDR
VL 7C6 CDR-L1RSTQTLVHRNGDTYLE:
Residues 24-39 of SEQ ID NO:12
VL 7C6 CDR-L2KVSNRFS:
Residues 55-61 of SEQ ID NO:12
VL 7C6 CDR-L3FQGSHVPYT:
Residues 94-102 of SEQ ID NO:12
VH 4B7 set CDR
VH 4B7 CDR-H1DYEMV:
Residues 31-35 of SEQ ID NO:15
VH 4B7 CDR-H2YISSGSRTIHYADTVKG:
Residues 50-66 of SEQ ID NO:15

VH 4B7 CDR-H3TLLRLHFDY:
Residues 99-107 of SEQ ID NO:15
VL 4B7 set CDR
VL 4B7 CDR-L1RSSQSLFYRSNQKNFLA:
Residues 24-40 of SEQ ID NO:16
VL 4B7 CDR-L2WASTRES:
Residues 56-62 of SEQ ID NO:16
VL 4B7 CDR-L3QQYYSYPWT:
Residues 95-103 of SEQ ID NO:16
VH 2F2 set CDR
VH 2F2 CDR-H1TFYIH:
Residues 31-35 of SEQ ID NO:19
VH 2F2 CDR-H2MIGPGSGNTYYNEMFKD:
Residues 50-66 of SEQ ID NO:19
VH 2F2 CDR-H3AKSARAAWFAY:
Residues 99-109 of SEQ ID NO:19
VL 2F2 set CDR
VL 2F2 CDR-L1RSSQSVVQSNGNTYLE:
Residues 24-39 of SEQ ID NO:20
VL 2F2 CDR-L2KVSNRFS:
Residues 55-61 of SEQ ID NO:20
VL 2F2 CDR-L3FQGSHVPPT:
Residues 94-102 of SEQ ID NO:20
VH 6A2 set CDR
VH 6A2 CDR-H1TFYIH:
Residues 31-35 of SEQ ID NO:23
VH 6A2 CDR-H2MIGPGSGNTYYNEMFKD:
Residues 50-66 of SEQ ID NO:23
VH 6A2 CDR-H3AKSHRAAWFAY:
Residues 99-109 of SEQ ID NO:23
VL 6A2 set CDR
VL 6A2 CDR-L1RSSQSVVQSNGNTYLE:
Residues 24-39 of SEQ ID NO:24

VL 6A2 CDR-L2
KVSNRFF:
Residues 55-61 of SEQ ID NO:24
VL 6A2 CDR-L3FQGSHVPPT:
Residues 94-102 of SEQ ID NO:24
VH 4D10 set CDR
VH 4D10 CDR-H1SYGVH:
Residues 31-35 of SEQ ID NO:27
VH 4D10 CDR-H2VIWRGGRIDYNAAFMS:
Residues 50-65 of SEQ ID NO:27
VH 4D10 CDR-H3NSDV:
The remains 98-101 of SEQ ID NO:27
VL 4D10 set CDR
VL 4D10 CDR-L1KSSQSLLDIDGKTYLN:
Residues 24-39 of SEQ ID NO:28
VL 4D10 CDR-L2LVSKLDS:
Residues 55-61 of SEQ ID NO:28
VL 4D10 CDR-L3WQGTHFPYT:
Residues 94-102 of SEQ ID NO:28
VH 7E5 set CDR
VH 7E5 CDR-H1DYEMV:
Residues 31-35 of SEQ ID NO:31
VH 7E5 CDR-H2YISSGSRTIHYADTVKG:
Residues 50-66 of SEQ ID NO:31
VH 7E5 CDR-H3TLLRLHFDY:
Residues 99-107 of SEQ ID NO:31
VL 7E5 set DR
VL 7E5 CDR-L1RSSQSLFYRSNQKNFLA:
Residues 24-40 of SEQ ID NO:32
VL 7E5 CDR-L2WASTRES:
Residues 56-62 of SEQ ID NO:32
VL 7E5 CDR-L3QQYYSYPWT:
Residues 95-103 of SEQ ID NO:32
VH 10C1 set CDR

VH 10C1 CDR-H1DYEMV:
Residues 31-35 of SEQ ID NO:35
VH 10C1 CDR-H2YINSGSGTIHYADTVKG:
Residues 50-66 of SEQ ID NO:35
VH 10C1 CDR-H3TLLRLHFDY:
Residues 99-107 of SEQ ID NO:35
VL 10C1 set CDR
VL 10C1 CDR-L1KSSQSLFYSRNQKNFLA:
Residues 24-40 of SEQ ID NO:36
VL 10C1 CDR-L2WASTGES:
Residues 56-62 of SEQ ID NO:36
VL 10C1 CDR-L3QQYFSYPWT:
Residues 95-103 of SEQ ID NO:36
VH 3B10 set CDR
VH 3B10 CDR-H1DYVIH:
Residues 31-35 of SEQ ID NO:38
VH 3B10 CDR-H2YINPYNDGTQYNEKFKG:
Residues 50-66 of SEQ ID NO:38
VH 3B10 CDR-H3VEGGTWDGYFDV:
Residues 98-109 of SEQ ID NO:38

In one embodiment, the antibody according to the invention contains at least two sets of CDR variable domains. More preferably, two sets of CDR variable domains selected from the group consisting of: CDR set VH 5F7 and CDR set VL 5F7; CDR set VH 10F11 and CDR set VL 10F11; CDR set VH 7C6 and CDR set VL 7C6; CDR set VH 4B7 and CDR set VL 4B7; CDR set VH 2F2 and CDR set VL 2F2; CDR set VH 6A2 and CDR set VL 6A2; CDR set VH 4D10 and CDR set VL 4D10; CDR set VH 7E5 and CDR set VL 7E5; and CDR set VH 10C1 and CDR set VL 10C1.

In another embodiment, the antibody described above, contains the human acceptor framework region.

In a preferred embodiment, the antibody is a CDR-grafted antibody. Preferably, the CDR-grafted antibody contains one or more CDRs described above.

Preferably, the CDR-grafted antibody contains a human acceptor framework region.

In a preferred embodiment, the antibody is humanitariannet antibody. Preferably, humanitariannet antibody contains one or more CDRs described above. More preferably, humanitariannet antibody contains tiili more CDR, described above. Most preferably, humanitariannet antibody contains six CDRs described above. In a specific embodiment, the CDR is embedded in the variable domain of the human acceptor framework region of human antibodies. Preferably, the variable domain of human antibodies is a consensus human variable domain. More preferably, the human acceptor framework region contains at least one substitution of key amino acid residue of the frame area where the key residue selected from the group consisting of a residue adjacent to a CDR; the rest of the glycosylation site, a rare residue; a residue capable of interacting with globularia Aβ(20-42); residue capable of interacting with a CDR; a canonical residue; residue from the contact area between the variable region of the heavy chain and the variable region of light chain; residue within a Vernier zone; the remainder is in the area of overlapping between the variable CDR1 of the heavy chain, defined by Chothia and the first frame region of the heavy chain, defined by Kabat. Preferably, the human acceptor framework region contains at least one substitution of amino acids in the framework region, where the amino acid sequence of the framework is at least 65% identical to the sequence specified AK is atomnogo frame and contains at least 70 amino acid residues, identical to the specified human acceptor framework region.

In an additional aspect, the present invention relates to antibodies containing both heavy and light chain as defined above.

Preferably, the antibody comprises at least one variable domain having the amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:38. More preferably, the antibody contains two variable domain, where the two variable domain have amino acid sequences selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:8 and SEQ ID NO:11 and SEQ ID NO:12, SEQ ID NO:15 and SEQ ID NO:16, SEQ ID NO: 19 and SEQ ID NO:20, SEQ ID NO:23 and SEQ ID NO:24, SEQ ID NO:27 and SEQ ID NO:28, SEQ ID NO:31 and SEQ ID NO:32, and SEQ ID NO:35 and SEQ ID NO:36.

In another aspect, antibodies of the present invention contain a constant region of the heavy chain selected from the group consisting of the constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and mutant Ala234 Ala235 human IgG1. In particular, the antibody contains a constant region of a human. More preferably, the antibody containing the amino acid sequence selected from the group consisting of SEQ ID nos:39-42. Preferred are antibodies containing constant is blast heavy chain IgG1.

In another embodiment, the antibody is glycosylated. Preferably, the pattern of glycosylation is the nature of the glycosylation of a person or the nature of the glycosylation obtained in any of eukaryotic cells described here, in particular in CHO cells.

The present invention relates also to antigennegative part of the antibodies of the present invention. Such antigennegative part include as non-limiting examples, Fab fragments, F(ab')2fragments and single-chain Fv fragments of antibodies. Additional antigennegative part is a Fab'fragments, Fv fragments, and linked by disulfide Fv-fragments.

The invention relates also to a selected nucleic acid encoding any of the antibodies described herein. Additional variant of implementation refers to the vector containing the selected nucleic acid described herein. The specified vector may, in particular, be selected from the group consisting of pcDNA; pTT (Durocher et al., Nucleic Acids Research 2002, Vol 30, No.2); pTT3 (pTT with additional polylinker; pEFBOS (Mizushima, S. and Nagata, S., (1990) Nucleic Acids Research Vol 18, No. 17); pBV; pJV; and a Shader with a.

In another aspect, the cell host transformed with the vector described herein. Preferably, a host cell is a prokaryotic cell. More prefer is Ino, a host cell is an E. coli. In a related embodiment, a host cell is a eukaryotic cell. Preferably, the eukaryotic cell is selected from the group consisting of cell protist, animal cells (such as mammalian cells, cells of birds and insect cells), plant cells and fungi cells. More preferably, a host cell is a mammalian cell, including, as non-limiting examples, CHO and COS; or cell fungi, for example, a yeast cell such as Saccharomyces cerevisiae; or an insect cell such as Sf9.

Another aspect of the invention relates to a method for producing antibodies according to the invention, comprising culturing any of the host cells or hybridoma described here, in a culture medium under conditions suitable for the production of antibodies. Another variant of implementation refers to the antibody that can be obtained in the manner described here.

Antibodies of the present invention can be obtained by a method known in essence.

B-lymphocytes, which generally contain a repertoire of antibodies composed of hundreds of billions of different specificdate antibodies are part of the immune system of mammals. The normal immune response to a specific antigen means selecting one or more antibodies from the specified repertoi is a, which are specifically associated with the specified antigen, and a successful immune response is based at least in part, on the ability of these antibodies specifically recognize (and ultimately remove) stimulating antigen and ignore other molecules surrounded by these antibodies.

The applicability of antibodies that specifically recognize one specific antigen target, led to the development of technologies of monoclonal antibodies. A standardized way hybrid currently allows obtaining antibodies with a single specificity to a specific antigen. Later methods have been developed for recombinant antibodies, such as the screening of libraries of antibodiesin-vitro. Similarly, these methods allow to obtain antibodies with a single specificity to a specific antigen.

In the method according to the invention the antigen of interest can be allowed to influence the repertoire of antibodies orin vivoorin vitro.

According to one variant of implementation of the antigen allow you to work on repertoire by immunizing an animal specified antigenin vivo. This methodin vivocan also include a number of hybrid of the lymphocytes of the animal and the selection of specific hybridoma, secreting the antibody specifically binding with the specified antigen. The stomach is passed, subject immunization may represent, for example, mouse, rat, rabbit, chicken, camel or lamb, or may be a transgenic variant of any of the above animals, for example, transgenic mice with the genes of the human immunoglobulin producing human antibodies after antigenic stimulus. Other types of animals that can immunize include mice with severe combined immunodeficiency (SCID), which was restored with the help of mononuclear cells of peripheral blood (chimeric mouse hu-PBMC SCID) or using lymphoid cells or their precursors, as well as mice that were treated with a lethal dose of radiation, then protected against exposure to bone marrow cells from mice with severe combined immunodeficiency (SCID), followed by transplantation of functional human lymphocytes ("Trimer"). Another type of animal that is subjected to immunization is an animal (e.g. a mouse), the genome of which the endogenous gene encoding the desired antigen, off (nokautiruet), for example, by homologous recombination, so that after immunization with antigen specified animal recognizes the specified antigen as foreign. The person skilled in the art it is obvious that polyclonal or monoclonal antibodies, polucen is E. in this way, characterize and select using known methods of screening that include as non-limiting examples of methods of ELISA and dot blot.

According to another variant implementation of the antigen allow you to work on the repertoire of antibodiesin vitroby screening libraries of recombinant antibodies using the specified antigen. Recombinant library of antibodies can Express, for example, on the surface of bacteriophages, or on the surface of yeast cells, or on the surface of bacterial cells. In many embodiments, the library of recombinant antibodies represents, for example, a library of scFv or Fab library. According to another variant implementation of the library of antibodies Express as a fusion RNA-proteins.

Another method of producing antibodies according to the invention includes a combination of approachesin vivoandin vitro. For example, the antigen can be allowed to influence the repertoire of antibodies by immunization of an animal specified antigenin vivosubsequent screening ofin vitrousing the specified antigen libraries of recombinant antibodies derived from lymphoid cells of the specified animal or library of single domain antibodies (e.g., containing heavy and/or light chain). According to another method the antigen to pozvoljaetrealizovat on the repertoire of antibodies by immunization of an animal specified antigen in vivoand then subjected to a library of recombinant antibodies or library separate domain derived from lymphoid cells of the specified animal affinity maturation. According to another method the antigen allow you to work on the repertoire of antibodies by immunization of an animal specified antigenin vivothen selection of producing antibodies cells secreting antibody of interest, receipt of these selected cells with cDNA for the variable regions of the heavy and light chains (e.g., PCR methods), and expression of the indicated variable regions of the heavy and light chains in the cells of the host mammalin vitro(this is indicated by the antibodies of the selected lymphocyte or SLAM), thereby obtaining the opportunity for further selection of the selected gene sequences of antibodies and manipulating them. Moreover, monoclonal antibodies can be selected expression cloning by gene for the heavy and light chains of antibodies in mammalian cells and selection of mammalian cells secreting antibody having the desired binding affinity of.

The present invention relates to certain antigens for screening and contrcting. Thus, it is possible according to the invention, to select such polyclonal and m is nacionalnye antibodies associated with globularia Aβ(20-42) affiniscape binding as defined above.

The methods according to the invention for obtaining antibodies can be used to obtain different types of antibodies. They include monoclonal, in particular recombinant antibodies, especially in native human antibodies, chimeric antibodies, humanized antibodies and CDR-grafted antibodies, as well as their antigennegative part.

In addition, the present invention relates to hybridoma, capable of producing (secrete) monoclonal antibody of the present invention. Hybridoma of the present invention include hybridoma outlined in the American type culture collection number Deposit selected from the group consisting of PTA-7241, PTA-7239, PTA-7240, PTA-7242, PTA-7408, PTA-7409, PTA-7405, PTA-7809, PTA-7810 and PTA-7851.

It is noted that the antibodies of the present invention can also be reactive, i.e. contact form Aβ, other than globalmenu Aβ described here. These antigens may or may not be oligomeric or globularity. Thus, the antigens that bind antibodies of the present invention, include any form of Aβ containing the epitope of globularia for which the antibodies of the present invention are reactive. Such forms of Aβ, including the indicate in itself truncated and not truncated forms of Aβ(X-Y) (with X and Y, defined as above), such as forms of Aβ(20-42), Aβ(20-40), Aβ(12-42), Aβ(12-40), Aβ(1-42) and Aβ(1-40), provided that these forms contain the epitope of globularia.

The present invention also relates to compositions containing an antibody according to the invention or its antigennegative part, as defined above.

According to a particular variant of implementation of the specified composition is a pharmaceutical composition comprising the antibody according to the invention or antigennegative part and a pharmaceutically acceptable carrier.

The antibody according to the invention or antigennegative part, as defined above, preferably is able to neutralize, asin vitroandin vivoactivity globular Aβ or its derivative, with which it is associated. The indicated antibody or antigennegative part, thus, can be used for inhibiting the activity specified globular or its derivative, for example, in the preparation containing the specified globular or its derivative, or individuals-humans, or other mammals are specified globular or its derivative.

According to one of embodiments the invention relates to a method of inhibiting the activity specified globular, or its derivative, where the method includes the providing of the tee for antibodies according to the invention or its antigennegative side effects on globular, or its derivative, so that they inhibit the activity specified globular or its derivative. Specified activity can inhibit, for example,in vitro. For example, the antibody according to the invention or its antigennegative part can be added to the drug, such as a sample obtained from the subject or culture of cells that contain, or are suspected to contain, the specified globular or its derivative to inhibit the activity specified globular or its derivative in a specified sample. Alternatively, the activity of globular or its derivative can inhibit an individualin vivo.

Thus, in addition, the present invention relates to the use of antibodies or antigennegative part, as defined above, to obtain a pharmaceutical composition for the treatment or prevention of amyloidosis, in particular of amyloidosis is selected from the group consisting of Alzheimer's disease and amyloidosis with down syndrome. One of the aspects specified application according to the invention, therefore, is a method of treatment or prevention of amyloidosis, in particular Alzheimer's disease or amyloidosis with down syndrome, in need thereof of a subject, which includes an introduction to the subject antibodies or antigennegative part, as defined above. Use specified the CSOs antibodies or antigennegative parts for treatment and more importantly, prevention of amyloidosis, in particular with Alzheimer's disease or amyloidosis with down syndrome, is intended in particular for passive immunization. Accordingly, in the method of treatment or prevention of amyloidosis, in particular Alzheimer's disease or amyloidosis with down syndrome in need of this subject, one of the purposes of introducing the antibodies or antigennegative part of the subject is passive immunization of a subject against amyloidosis, in particular Alzheimer's disease or amyloidosis with down syndrome.

The antibody according to the invention or antigennegative part, as defined above, preferably, are suitable for detecting, asin vitroandin vivo, globular Aβ or its derivative, with which they are associated. The indicated antibody or antigennegative part, thus, can be used for detection of the specified globular or its derivative, for example, in the preparation containing the specified globular or its derivative, or individuals-humans, or other mammals are specified globular or its derivatives.

According to one of embodiments the invention relates to a method of detecting the specified globular or its derivative, where the method includes providing the antibody according to the invention or the th antigennegative part to influence globular or its derivative, so they contact the designated globularia or its derivatives (and thus preferably form a complex containing the antibody or antigennegative part and globular or its derivative). The specified globular can be used to detect, for example,in vitro. For example, the antibody according to the invention or antigennegative part can be added to the drug, such as a sample obtained from a subject or cell culture containing, or suspected of containing the specified globular or its derivative, for detection of the specified globular or its derivative in the specified drug. Alternatively, globular or its derivative can be used to detect the individualin vivo.

Thus, in addition, the present invention relates to the use of antibodies or antigennegative part, as defined above, to obtain a composition for the diagnosis of amyloidosis, in particular Alzheimer's disease or amyloidosis with down syndrome. One aspect of the specified application according to the invention is a method of diagnosis of amyloidosis, in particular Alzheimer's disease or amyloidosis with down syndrome, the subject allegedly suffering from amyloidosis, in particular Alzheimer's disease or the amyloidosis with down syndrome, comprising the administration to a subject the antibody or antigennegative the part, as defined above, and the detection of the formation of the complex containing the antibody or antigennegative part to the antigen, where the presence of the complex indicates amyloidosis, in particular Alzheimer's disease or the amyloidosis with down syndrome in the subject. The second aspect of the specified application according to the invention is a method for the diagnosis of amyloidosis, in particular Alzheimer's disease or amyloidosis with down syndrome, the subject allegedly suffering from amyloidosis, in particular Alzheimer's disease or the amyloidosis with down syndrome, comprising obtaining a sample from the subject, contacting the sample with the antibody or antigennegative part, as defined above, and the detection of the formation of the complex containing the antibody or antigennegative part to the antigen, where the presence of the complex indicates amyloidosis, in particular Alzheimer's disease or the amyloidosis with down syndrome in the subject.

Detailed description of the invention

The affinity of binding of the antibodies according to the invention can be assessed using standardized immunoassaysin vitrosuch as ELISA assays, dot blot or BIAcore (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). Further descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277.

p> According to a particular variant of implementation of the affinity, defined here refer to values obtained by carrying out the dot-blotting as described in example 8, and evaluating it by densitometry. According to a particular variant implementation of the invention the determination of the affinity of binding by dot-blotting includes the following: a specific amount of antigen (e.g., globular Aβ(X-Y), monomer Aβ(X-Y) or fibrils Aβ(X-Y), as defined above) or appropriate, their respective breeding, for example, 20 mm NaH2PO4, 140 mm NaCl, pH of 7.4, 0.2 mg/ml BSA to a concentration of antigen, for example, 100 pmol/µl 10 pmol/µl, 1 pmol/μl, 0.1 pmol/μl and 0.01 pmol/μl, put points on the nitrocellulose membrane, then the membrane blocking the milk to prevent nonspecific binding and washed, then put in contact with the target antibody, followed by detection of the latter by conjugated with the enzyme secondary antibody and colorimetric reaction; when certain concentrations of antibodies the amount of bound peroxidase antibodies allows determination of affinity. Thus, the relative affinity of the two different antibodies to the same target or one antibody for two different targets define here as the ratio of the corresponding quantities associated with isent antibodies observed for the two combinations of antibody-targeted in otherwise identical conditions the dot-blotting. Unlike similar way, based on Western-blotting according to the method of the dot-blotting will determine the affinity of the antibody for the target when the natural conformation of the latter; in contrast to the way ELISA method the dot-blotting does not suffer from differences in offendeth between different targets and the matrix, thus allowing more accurate comparisons between different targets.

The term "Kd"as used here is intended to denote the dissociation constants for the specific interaction of the antibody-antigen, as is well known in this field.

Antibodies of the present invention preferably are selected antibodies. "Isolated antibody" means an antibody having affiniscape binding, as described above, and mostly free of other antibodies having excellent affiniscape binding. The term "mostly free" here refers to the drug antibodies in which at least 95% of the antibodies, preferably, at least 98% of the antibodies and, more preferably, at least 99% of the antibodies have the desired binding affinity of. Moreover, the selected antibody may be essentially free of other cellular material and/or chemically the substances.

The selected antibodies according to the present invention include monoclonal antibodies. "Monoclonal antibody", as used here, is intended to refer to a preparation of antibody molecules, antibodies which share amino acid sequence of the total heavy chain and a common light chain, unlike drugs "polyclonal" antibodies containing a mixture of antibodies of different amino acid sequences. Monoclonal antibodies can be obtained through several new methods, such as phage, bacterial, yeast or ribosomal display, as well as classical methods, illustrated derived from hybridoma antibodies (e.g., antibody, secretively hybridomas obtained by the hybrid method, such as conventional hybrid method Kohler and Milstein ((1975) Nature 256:495-497). Thus, the antibody obtained not from hybridoma, with homogeneous sequence, still denoted here as a monoclonal antibody, although it can be obtained through non-classical methods, and the term "monoclonal" do not limit obtained from hybridoma antibodies, but is used as referring to all antibodies derived from a single clone nucleic acids.

Thus, the monoclonal antibodies of the present invention include recombinantly the antibodies. The term "recombinant" here refers to any artificial Union of two otherwise separated fragments of the sequence, for example, by chemical synthesis or by the manipulation of the selected nucleic acid fragments by means of genetic engineering. In particular, the term "recombinant antibody" refers to antibodies that produce, Express, receive or emit recombinant means, such as antibodies that Express using recombinant expressing vector, transfitsirovannykh in cell host; antibodies isolated from a recombinant, combinatorial libraries of antibodies; antibodies isolated from an animal (e.g. a mouse)that is transgenic for genes of human immunoglobulin (see, for example, Taylor, L.D., et al. (1992) Nucl. Acids Res. 20:6287-6295); or antibodies that produce, Express, receive or emit any other way in which specific sequences of immunoglobulin genes (such as gene-sequences of human immunoglobulins) are combined with other DNA sequences. Recombinant antibodies include, for example, chimeric, CDR-grafted, and humanized antibodies. Specialists in this field are aware of that for expression of monoclonal antibodies derived from a common hibrido is, in heterologous system must obtain recombinant antibodies, even if the amino acid sequence of the obtained protein-antibodies do not change or do not intend to change.

In a specific embodiment of the invention the antibody is humanitariannet antibody.

According to many embodiments the antibody may contain amino acid sequence, obtained from different species, such as human antibody or antibody mouse. According to other variants of implementation, the antibody may be a chimeric antibody or a CDR-grafted antibody, or another form gumanitarnogo antibodies.

The term "antibody" is intended to refer to immunoglobulin molecules, composed of 4 polypeptide chains, two heavy (H) chains and two light (L) chains. The circuit is generally connected to each other by disulfide bonds. Each heavy chain consists of a variable region of the specified heavy chain (designated here as HCVR or VH) and a constant region specified heavy chain. The constant region of the heavy chain consists of three domains, CH1, CH2 and CH3. Each light chain consists of a variable region of the specified light chain (designated here as LCVR or VL) and a constant region of the specified light chain. The constant region of the light chain consists of the domain CL. Region VH and VL can be further divided into hypervariable regions marked complementarity determining regions (CDR) and alternating conservative areas indicated frame regions (FR). Each VH and VL region, therefore, is composed of three CDRs and four FR, arranged from N-Terminus to the C-end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to specialists in this field.

The term "antigennegative part of the antibody (or simply "antibody"refers to one or more fragments of the antibodies according to the invention, where the specified portion(s) still has affiniscape binding as defined above. It is shown that fragments of full antibodies capable of antigennegative the function of antibodies. In accordance with the term "antigennegative part of the antibody, examples of binding fragments include (i) a Fab fragment, i.e. a monovalent fragment consisting of the VL domains, VH, CL and CH1; (ii) F(ab')2the fragment, i.e. a bivalent fragment comprising two Fab fragments linked with each other in the hinge region via a disulfide bridge; (iii) a Fd fragment consisting of the VH domains and CH1; (iv) an Fv fragment consisting of domains, FL and VH one shoulder antibodies; (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain or VH, CH1, CH2, DH3, or VH, CH2, CH3; and (vi) selected to define the expansion of the complementary region (CDR). Although the two domains of Fv-fragment, namely, VL and VH, are coded separate genes, they can optionally be connected to each other using a synthetic linker, for example amino acid sequence of poly-G4S, and recombinant methods, allowing to receive them as a single protein chain in which the VL region and a VH brought together to form monovalent molecules (known as single-chain Fv (ScFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). The term "antigennegative part of the antibody is also intended to include single-chain antibodies. Other forms of single-chain antibodies, such as "diately" likewise included here. Diately represent a bivalent, bespecifically antibodies, in which the domains VH and VL expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigenspecific plot (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). Constant domain of immunoglobulin refers to the constant domain of the heavy or light chain. Amino acid sequence of the constant the aqueous domain of the heavy chain and light chain of human IgG is known in this field and are presented in table 1.

Moreover, the antibody of the present invention or its antigennegative part can be part of a larger molecule of immunoadhesin, formed by covalent or non-covalent Association of the indicated antibodies or parts of antibodies with one or more proteins or peptides. Appropriate for such molecules immunoadhesin are using streptavidine cow area to obtain a tetramer of scFv molecules (Kipriyanov, S. M., et al. (1995)Human Antibodies and Hybri - domas6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine, for example exegetically labels to obtain bivalent and biotinylated scFv molecules (Kipriyanov, S.M., et al. (1994) Mol. Immunol. 31:1047-1058).

The term "human antibody" refers to antibodies, variable and constant region which correspond to the sequences of immunoglobulin germline human or received from them, as described, for example, Kabat et al. (see Kabat, et al. (1991) Sequences of Protein of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). However, human antibodies of the invention may include amino acid residues not encoded by the sequences of immunoglobulin germ line person (e.g., mutations introduced by random or site-SP is specific mutagenesis in vitroor by somatic mutationin vivo), for example in the CDRs and in particular CDR3. Recombinant human antibodies of the invention have variable regions and may also contain a constant region derived from immunoglobulin sequences of the germline of the person (see Kabat, E. A., et al. (1991) Sequences of Protein of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). However, according to a specific implementation options, such recombinant human antibodies are subjected to mutagenesisin-vitro(or somatic mutagenesisin-vivoif you use an animal transgenic for Ig sequences person), so that the amino acid sequence regions VH and VL of the recombinant antibodies are sequences that, while related to the sequences of VH and VL of the germ line human or derived from them, do not exist in naturein vivoin the repertoire of antibody germline person. According to specific variants of implementation of the recombinant antibodies of this type are the result of selective mutagenesis, or reverse mutation, or both. Preferably, the mutagenesis leads to the affinity to the target, which exceeds the affinity of the original antibody, and/or affinity to non-target structures, which is lower Affi the activity of the original antibody.

The term "chimeric antibody" refers to antibodies that contain sequences of variable regions of heavy and light chains of one type and sequence of the constant regions from other species, such as antibodies having variable regions of the heavy and light chain of the mouse associated with the constant regions of a human.

The term "CDR-grafted antibody" refers to antibodies that contain sequences of variable regions of heavy and light chain from one species but in which the sequences of one or more CDR regions of VH and/or VL are replaced with CDR sequences from other species, such as antibodies having variable regions of the heavy and light chains, in which one or more CDRs of the mouse (e.g., CDR3) are replaced with CDR sequences of a person.

The term "humanitariannet antibody" refers to antibodies that contain sequences of variable regions of heavy and light chains of the non-human species (e.g. mouse, rat, rabbit, chicken, camel, sheep or goats), in which, however, at least one part of the sequence of the VH and/or VL change, so they were more "human-like", i.e. were more similar to the variable sequences of the germline of the person. One type gumanitarnogo antibody is a CDR-when itoe antibody in which the sequence of CDR person inserted in non-human sequences of VH and VL to replace the corresponding non-human CDR sequences.

The terms "Kabat numbering", "definitions of Kabat and marking Kabat" are used here interchangeably. These terms, which are known in this field, refer to the system of numbering amino acid residues which are more variable (i.e. hypervariable)than other amino acid residues in the variable regions of the heavy and light chains of the antibody or its antigennegative part (Kabat et al. (1971) Ann. NY Acad, Sci 190:382-391 and Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). In the case of variable regions of the heavy chain hypervariable region is in the range of positions of the amino acids from 31 to 35 for CDR1, the provisions of amino acids 50 to 65 for CDR2, and the provisions of amino acids 95 to 102 for CDR3. In the case of variable regions of the heavy chain hypervariable region is in the range of positions of the amino acids 24 to 34 for CDR1, the provisions of amino acids 50 to 56 for CDR2, and the provisions of amino acids from 89 to 97 for CDR3.

As used here, the terms "acceptor" and "acceptor antibody" refers to a sequence of the antibody or nucleic acid representing or encoding at the ore 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% amino acid sequence of one or more frame regions. In some embodiments, the implementation of the term "acceptor" refers to the amino acid sequence of the antibody or nucleic acid sequence representing or encoding the constant region(oblast). In another embodiment, the term "acceptor" refers to the amino acid sequence of the antibody or nucleic acid sequence representing or encoding one or more frame regions and constant region(oblast). In a specific embodiment, the term "acceptor" refers to the amino acid sequence of human antibodies or nucleic acid sequence representing or encoding at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or 100% amino acid sequence of one or more frame regions. According to this variant of implementation, the acceptor may contain at least 1, at least 2, at least 3, at least 4, at least 5 or at least 10 amino acid residues not present in one or more specific provisions of the human antibodies. And zapornuju frame region and/or acceptor constant area(s), for example, take or receive from germline antibody gene, Mature gene antibodies, antibodies (e.g. antibodies, are well known in this field, developed antibodies or commercially available antibodies).

As used here, the term "CDR" refers to the complementarity determining region within the variable sequences of the antibodies. There are three CDRs in each of the variable regions of the heavy chain and light chain, designated CDR1, CDR2 and CDR3 for each variable regions. The term "CDR set", as used here, refers to a group of three CDRs present in one variable region capable of binding an antigen. The exact boundaries of these CDR define different according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous numbering system balances applicable to any variable regions of antibodies, but also provides precise boundaries of the residues that define the three CDR. These CDRs can be described Kabat CDR. Chothia and co-authors (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that the specific subfragment inside Kabat CDR accept almost identical conformation of the peptide backbone, despite the great diversity at the level of amino acid sequence. These subfragment about who appoints L1, L2 and L3 and H1, H2 and H3, where "L" and "H" indicate the region of the light chain and heavy chains, respectively. These areas can be described Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDR, overlapping with the Kabat CDRs, defined Padlan (FASEB J. 9:pages 133-139 (1995)) and MacCallum (J. Mol. Biol. 262(5):732-45 (1996)). Other definitions of the boundaries of the CDR may not strictly follow one of the above systems, however, will overlap with the Kabat CDRs, although they may be shortened or lengthened in the light of predictions or experimental discovery that specific residues or groups of residues or even entire CDR does not significantly contribute to the binding to the antigen. The methods used here can be used CDRs, as defined according to any one of these systems, although in preferred embodiments, the implementation of the use of CDRs defined according to Kabat or Chothia.

As used here, the term "canonical" balance refers to the residue in a CDR or framework, which defines the particular canonical CDR structure as defined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992), both listed here as references). According to Chothia et al. the critical part of the CDR of many antibodies have nearly identical conformations of the peptide backbone in spite of the great diversity at the level of amino acid sequence. Each the I canonical structure specifies a first set of torsion angles of the peptide backbone for continuous fragment of the amino acid residues, forming a loop.

As used here, the terms "donor" and "donor antibody" refers to an antibody that provides one or more CDR. In a preferred embodiment, the donor antibody is an antibody from a species other than the species for antibody from which you obtained or derived frame area. In the context of gumanitarnogo antibodies, the term "donor antibody" refers to non-human antibody, providing one or more CDR.

As used here, the term "frame" or "frame sequence" refers to the remaining sequences of the variable regions minus CDR. Because the exact set boundaries CDR sequence can be defined using different systems, the value of the frame sequence is subject to different interpretations. Six CDRs (CDR-L1, -L2 and-L3 of the light chain and CDR-H1, -H2, and-H3 of the heavy chain) also share the frame region of the light chain and heavy chain four subfragment (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without identifying specific subfragments as FR1, FR2, FR3 or FR4 frame area, as determined by others, is a United FR variable inside the scope of individual, existing in the nature of chain immunoglob is Lina. As used here, FR is one of the four subfragments, and FR plural represents two or more of the four subfragments constituting a frame area.

Acceptor sequence of the heavy chain and light chain of a man known in this field.

As used here, the term "germline antibody gene" or "gene fragment" refers to a sequence of immunoglobulin encoded by non-lymphoid cells that had not undergone the maturation process, leading to genetic rearrangeable and mutation for expression of specific immunoglobulin (see, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchlnis et al., Adv Exp Med Biol. 484:13-30 (2001)). One of the advantages provided by different variants of implementation of the present invention stems from the discovery that germline antibody genes are more likely than Mature genes antibodies retain the desired structure amino acid sequence characteristic of individuals in the species, thus, less likely recognizable as its not when used in these types.

As used here, the term "key" residues refers to specific residues within the variable regions that contribute more to the binding specificity and/or affinity of antibodies, in particular gumanitarnogo antibodies. To achevie residues include, without limitation, one or more of the following: a residue adjacent to a CDR, a potential glycosylation site (which may represent an area or an N-or O-glycosylation), a rare residue, a residue capable of interacting with antigen, a residue capable of interacting with a CDR, a canonical residue, a residue of the contact area between the variable region of the heavy chain and the variable region of the light chain, residue within the Vernier zone, and a residue in a region of overlap between the defined by Chothia variable CDR1 of the heavy chain and the one defined by Kabat framework first heavy chain.

As used here, the term "humanitariannet antibody" refers specifically to the antibody or variant, derivative, analog or fragment, which immunospecificity associated with the antigen of interest and which contains the framework (FR) region, with mainly the amino acid sequence of human antibodies and complementarity determining region (CDR)having mainly amino acid sequence of the non-human antibody. As used here, the term "mainly" in the context of CDR refers to a CDR having the amino acid sequence at least 80%, preferably at least 85%, at least 90%, at least 95%, hence, is her least 98% or at least 99% identical to the amino acid sequence of CDR of non-human antibodies. Humanitariannet antibody contains basically all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv)in which all or substantially all of the CDR regions correspond to non-human immunoglobulin (i.e donor antibody) and all or substantially all of the frame areas represent the areas with the consensus sequence of human immunoglobulin. Preferably, humanitariannet antibody also contains at least part of a constant region (Fc) of an immunoglobulin, typically a region of human immunoglobulin. In some embodiments, the implementation humanitariannet antibody containing light chain and at least the variable domain of the heavy chain. The antibody may also contain CH1, hinge, CH2, CH3, and CH4 region of the heavy chain. In some embodiments, the implementation humanitariannet antibody contains only humanitarian light chain. In some embodiments, the implementation humanitariannet antibody contains only humanitarian heavy chain. In specific embodiments, the implementation humanitariannet antibody contains only humanitarianly variable domain light chain and/or humanitarian heavy chain.

Humanitariannet antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE,and any subclass, including, without limitation, IgG1, IgG2, IgG3 and IgG4.

Frame and CDR region gumanitarnogo antibodies is not necessarily exactly correspond to the original sequence, for example, CDR or the consensus framework donor antibodies can be subjected to mutagenesis by replacement, insertion and/or deletion of at least one amino acid residue so that the residue CDR or framework in this area does not match exactly any donor antibody or consensus framework. In a preferred embodiment, such mutations, however, will not be extensive. Usually at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% of the residues gumanitarnogo antibodies will meet the remnants of the original sequences of the FR and CDR. As used here, the term "consensus framework" refers to the frame region of the consensus sequence of the immunoglobulin. As used here, the term "consensus sequence of immunoglobulin" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences of immunoglobulins (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany, 1987). In the family of immunoglobulins each position in the consensus last the successive busy amino acid, the most common in this position in the family. When two amino acids occur with equal frequency, any can be included in the consensus sequence.

As used here, the area of the "Vernier" denotes the subgroup frame residues that can regulate the structure of the CDR and to fine-tune to match the antigen, as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, the contents of which are hereby incorporated by reference). The remains of the Vernier zone form the layer, the underlying CDR, and can contribute to the structure of the CDR and the affinity of antibodies.

The term "epitope" includes any protein determinant capable of specifically bind with an antibody. In specific embodiments, the implementation of the epitope determinants include chemically active surface groupings of molecules such as amino acids, the side chains of sugars, phosphoryl or sulfonyl, and, in specific embodiments, the implementation may have specific three dimensional structural characteristics, and/or specific charge characteristics. The epitope is a region of the antigen bound by the antibody. In specific embodiments, implementation indicate that the antibody specifically binds to the antigen, mainly when it recognizes its antigen target in a complex mixture of proteins and/or macro is Alakul.

The term "polynucleotide", as denoted here refers to a polymeric form of two or more nucleotides, either ribonucleotides or deoxynucleotides, or modified forms of any type nucleotides. The term includes single and double-stranded forms of DNA, but preferably the DNA is double-stranded DNA.

The term "isolated polynucleotide"as used here shall mean polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or any combination), such that, by virtue of its origin, "selected polynucleotide" is not associated with all or part of polynucleotide with which the "isolated polynucleotide found in nature; is functionally related polynucleotide with which it is associated in nature; or does not occur in nature as part of a larger sequence.

The term "vector"as used here is intended to denote a molecule of nucleic acid, capable of carrying another nucleic acid to which it is associated. One type of vector is a "plasmid", which refers to the circular loop of double-stranded DNA, which can be ligitamate additional DNA fragments. Another type of vector is a viral vector, where additional DNA fragments can be ligitamate in the viral genome. Specific vectors are SP is capable for Autonomous replication in a cell-master, in which they are introduced (e.g., bacterial vectors having a bacterial replication origin and episomal vectors mammals). Other vectors (e.g., episomal vectors mammals) can be integrated into the genome of a host cell upon introduction into the cell of the host, and thus they are replicated together with the genome of the host. In addition, specific vectors capable of driving the expression of genes with which they are functionally linked. Such vectors are "expressing recombinant vectors" (or simply "expressing vectors"). As a rule, expressing vectors for use in the methods of recombinant DNA is often present in the form of plasmids. In the present description "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly accepted form of the vector. However, the invention is intended to include other such forms expressing vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which perform equivalent functions.

The term "functionally linked" means the immediate neighborhood, where the components described are in a relationship permitting them to function in the intended way. Control the sequence, "functionally linked" to the coding sequence attached in such a way that expression of the coding sequence get in terms of appropriate control sequences. "Functionally related sequences include as controls the expression of the sequence adjacent to the gene of interest and controlling the expression of sequences that control gene of interest are in TRANS-position is obtained, or at a distance. The term "controlling the expression sequence", as used here, refers to polynucleotide sequences that are necessary to influence the expression and processing of coding sequences to which they legirovanyh. Controlling the expression sequences include appropriate sequences of transcription initiation, termination, promoter and enhancer; signals efficient RNA processing, such as splicing signals, polyadenylation; sequences that stabilize cytoplasmic mRNA; sequences that enhance the efficiency of translation (i.e. a consensus Kozak sequence); sequences that enhance protein stability; and when it is desirable, sequences that enhance protein secretion. The nature of such control pic is of egovernance differs depending on the host body; for prokaryotes, such control sequences generally include promoter, the binding site of the ribosome and the sequence termination of transcription; in eukaryotes, generally, such control sequences include a promoter and termination sequence transcription. The term "control sequences" is intended to include components whose presence is necessary for expression and processing, and may also include additional components whose presence is advantageous, for example, leader sequences and sequences of merger partner.

"Transformation", as defined here, refers to any method by which exogenous DNA enters the cell host. The transformation can be done in a natural or artificial conditions using various methods, well known in this field. The transformation can be based on any known method of introducing sequences alien nucleic acid in a prokaryotic or eukaryotic cell host. The method is chosen based on the subject of transformation of the host cell, and may include as non-limiting examples of viral infection, electroporation, lipofection and bombing is iruku particles. Such "transformed" cells include stably transformed cells in which the inserted DNA is able to replicate either as a stand-alone can replicate a plasmid or as part of a chromosome of the host. They also include cells, temporarily expressing the inserted DNA or RNA for limited periods of time.

The term "recombinant a host cell" (or simply "a host cell", as used here, is intended to refer to cells in which the introduced exogenous DNA. It should be understood that such terms are intended to refer not only to specific the cell, and the progeny of such cells. Because in subsequent generations may be certain modifications, as a result of either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included in the scope of the term "a host cell", as used here. Preferably, the cell hosts include prokaryotic and eukaryotic cells selected from any of the kingdoms of living organisms. Preferred eukaryotic cells include cells of protists, fungi, plants and animals. Most preferably, the cell hosts include as non-limiting examples of line prokaryotes the cells E. coli; lines of mammalian cells CHO, HEK 293 and COS; the line of insect cells Sf9; and the cells of fungiSaccharomyces cerevisiae.

Conventional methods can be used for recombinant DNA, synthesis of oligonucleotides, cell culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions, or as is common in this area, or as described herein. The above methods can basically be carried out according to conventional methods well known in the field, and as described in various General and more specific references that are cited and discussed throughout the present description. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), given here as a reference for all purposes.

"Transgenic organism", as it is known in this field, as used here, refers to an organism having cells that contain a transgene, where the transgene is introduced into the body (or into an ancestor of the organism), Express a polypeptide that is naturally expressed in the body. "Transgene" is a structure of DNA that is stably and functionally integrated into the host cell genome, from which it develops transgenic org the ISM, driving expression of an encoded gene product in one or more cell types or tissues of the transgenic organism.

Methods for producing antibodies according to the invention are described below. Made the separation between the waysin-vivo, howin-vitroor a combination of both.

Some methods for producing antibodies according to the invention are described below. Made the separation between the waysin-vivo, howin-vitroor a combination of both.

Howin-vivo

Depending on the type of the desired antibodies, various host animals can be used for immunizationin-vivo. You can use the master, who himself expresses endogenous variant antigen of interest. Alternatively, you can use host deficient in endogenous variant antigen of interest. For example, have shown that mice made deficient in specific endogenous protein by homologous recombination in the corresponding endogenous gene (i.e. knocked out mice) produce a humoral response to the protein to which they are subjected to immunization and thus they can be used to obtain high-affinity monoclonal antibodies to the protein (see, for example, Roes, J. et al. (1995) J. Immunol. Methods 183:231-237; Lunn, MP. et al. (2000) J. Neurochem. 75:404-412).

Many non-human mammals are suitable hosts for the production of the antibodies is to obtain a non-human antibody according to the invention. They include mice, rats, chickens, camels, rabbits, sheep and goats (and their knocked-out options), although I prefer mice to generate hybridomas. In addition, non-human animal host expressing a repertoire of human antibodies, can be used to obtain the necessary human antibodies with dual specificity. Non-human animals of this species include transgenic animals (e.g. mice)carrying the transgene of human immunoglobulins (chimeric mouse hu-PBMC SCID) and irradiated Chimera human/mouse, described in more detail below. According to one variant of implementation, the animal is immunized with globularia Aβ(20-42) or its derivative, is a non-human animal, preferably a mouse that is transgenic for genes of human immunoglobulins, so that the specified non-human mammal produces human antibodies with antigenic stimulation. As a rule, the transgenic immunoglobulin heavy and light chain configuration germline person impose such animals, which are modified so that their endogenous loci heavy and light chains are inactive. If such animals stimulate antigen (e.g., antigen person), they produce antibodies obtained from p is sledovatelnot immunoglobulin (human antibodies). Can be obtained from the cells of such animals monoclonal human antibodies through a standardized way of hybridoma. Additional description of transgenic mice with antibodies and their use for obtaining human antibodies, see, for example, in US 5939598, WO 96/33735 and WO 96/34096, WO 98/24893 and WO 99/53049 (Abgenix Inc.), and US 5545806, US 5569825, US 5625126, US 5633425, US 5661016, US 5770429, US 5814318, US 5877397 and WO 99/45962 (Genpharm Inc.); see also MacQuitty, J.J. and Kay, R. (1992) Science 257:1188; Taylor, L.D. et al. (1992) Nucleic Acid Res. 20:6287-6295; Lonberg, N. et al. (1994) Nature 368:856-859; Lonberg, N. and Huszar, D. (1995) Int. Rev. Immunol. 13:65-93; Harding, F.A. and Lonberg, N. (1995) Ann. N Y. Acad. Sci. 764:536-546; Fishwild, D. M. et al. (1996) Nature Biotechnology 14:845-851; Mendez, M. J. et al. (1997) Nature Genetics 15:146-156; Green, L.L. and Jakobovits, A. (1998; J. Exp. Med. 188:483-495; Green, L.L. (1999) J. Immunol. Methods 231:11-23; Yang, X.D. et al. (1999) J. Leukoc. Biol. 66:401-410; Gallo, M.L. et al. (2000) Eur. J. Immunol. 30:534-540.

According to another variant implementation of the animal, which is subjected to immunization with globularia Aβ(20-42) or its derivatives, may be a mouse with severe combined immunodeficiency (SCID), which was restored by mononuclear cells of peripheral blood or lymphoid cells or their precursors. Such mice, labeled chimeric mice hu-PBMC SCID been shown to produce responses of human immunoglobulins with antigenic stimulation. Additional description of these mice and their use to generate antibodies, see, for example, in Leader, K.A. et al (1992) Immunology 76:229-234; Bombil, F. et al. (1996) Immunobiol. 195:360-375; Murphy, W.J. et al. (1996) Semin. Immunol. 8:233-241; Herz, U. et al. (1997) Int. Arch. Allergy Immunol. 113:150-152; Albert, S.E. et al. (1997) J. Immunol. 159:1393-1403; Nguyen, H. et al. (1997) Environ. Immunol. 41:901-907; Arai, K. et al. (1998) J. Immunol. Methods 217:79-85; Yoshinari, K. and Arai, K. (1998) Hybridoma 17:41-45; Hutchins, W.A. et al. (1999) Hybridoma 18:121-129; Murphy, W.J. et al. (1999) Clin. Immunol. 90:22-27; Smithson, S. L. et al. (1999) Mol. Immunol. 36:113-124; Chamat, S. et al. (1999) J. Infect. Diseases 180:268-277; and Heard, C. et al. (1999) Molec. Med. 5:35-45.

According to another variant implementation of the animal, which is subjected to immunization with globularia Aβ(20-42) or its derivatives, is a mouse that was treated with a lethal dose of total body irradiation, and then protected from exposure to bone marrow cells from mice with severe combined immunodeficiency (SCID) and then transplanted functional human lymphocytes. This type chimeras indicated by the system trimer, were used to produce monoclonal antibodies through immunization of these mice antigen of interest and then get monoclonal antibodies through the use of a standardized method hybridoma. Additional description of these mice and their application to generate antibodies see, for example, Eren, R. et al. (1998) Immunology 93:154-161; Reisner, Y and Dagan petrol, S. (1998) Trends Biotechnol. 16:242 - 246; Nan, E. et al. (1999) Hepatology 29:553-562; and Bocher, W.O. et al. (1999) Immunology 96:634-641.

Since the obtainedin-vivoproducing antibody cells, monoclonalantibody can be obtained through standardized ways, such as the way hybridoma, originally described by Kohler and Milstein (1975, Nature 256:495-497) (see also Brown et al. (1981) J. Immunol 127:539-46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75). The method of obtaining hybridomas for monoclonal antibodies is widely known (see, generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet, 3:231-36). Briefly, immortalizing cell line (typically myeloma) is drained to lymphocytes (typically splenocytes or cells of lymph nodes or peripheral blood lymphocytes) of a mammal immunized with globularia Aβ according to the invention or its derivative, and supernatant culture derived cells hybridoma sceneroot to identify hybridoma producing a monoclonal antibody of the present invention. Any of a variety of well known methods for fusion of lymphocytes and immortalized cell lines can be applied for this purpose (see also, G. Galfre et al. (1977) Nature 266:550-52; Gefter et al. Somatic Cell Genet, cited above; Lerner, Yale J. Biol. Med., cited above; Kenneth, Monoclonal Antibodies, cited above). Moreover, an experienced specialist in this field will recognize that there are different versions of these methods that are applicable in a similar manner. As a rule, immorta sofanou cell line (for example, line of myeloma cells) are obtained from the same mammal species as the lymphocytes. For example, it is possible to stabilize the mouse hybridoma by fusion of lymphocytes from a mouse immunized with immunogenic preparation according to the invention, with immortalizing line of mouse cells. Preferred termed the cell lines are lines are murine myeloma cells that are sensitive to culture medium containing gipoksantin, aminopterin and thymidine (Wednesday HAT). Any one of a number of lines of myeloma cells can be used by default as a partner in the merger, for example, myeloma line P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14. These lines of myeloma cells available in the American type culture collection (ATCC), Rockville, MD. As a rule, HAT-sensitive myeloma cells mouse merge with mouse splenocytes using polyethylene glycol (PEG). Then cells hybridoma received in the merger, are selected by using a HAT, thus killing naslite and unproductively fused myeloma cells (naslite splenocytes die after several days because they are not transformed). Cell hybridoma producing monoclonal antibodies according to the invention, identified by screening supernatants culture hybridoma such antibodies, for example, through the use of analysis datamation, as described above in example 8, to take such antibodies, which have affiniscape binding as defined above.

Monoclonal antibodies 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 and 3B10 all obtained using the above described methodin-vivoand you can get them from hybridoma, as defined here.

Similarly, the specified hybridoma can be used as a source of nucleic acid that encodes a light and/or heavy chain, recombinant generate antibodies of the present invention, as described in more detail below.

Howin-vitro

Alternatively, the antibodies according to the invention through immunization and selection of antibodies according to the invention it is possible to identify and allocate by screening a recombinant combinatorial libraries of immunoglobulins using globular Aβ(20-42) or its derivative, so as to distinguish the members of a library of antibodies with desired binding affinity of. Kits for receiving and screening display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, catalog No. 27-9400-01; and the Stratagene SurfZAP® Phage Display Kit, catalog No. 240612). In many embodiments, the implementation of the library display is a library of scFv or Fab library. The method of phage display for screening libraries recombin ntih antibodies described sufficiently. Examples of methods and compounds that can be used particularly preferably for receiving and screening libraries display of antibodies can be found, for example, McCafferty et al. WO 92/01047, US 5969108 and EP 589877 (described, in particular, the display scFv), Ladner et al. US 5223409, US 5403484, US 5571698, US 5837500 and EP 436597 (described, for example, a protein with pIII); Dower et al. WO 91/17271, US 5427908, US 5580717 and EP 527839 (described, in particular, Fab display); Winter et al. International publication WO 92/20791 and EP 368684 (described, in particular, the cloning of sequences for the variable domains of immunoglobulins); Griffiths et al. US 5885793 and EP 589877 (described, in particular, the selection of human antibodies against antigens of the person using recombinant libraries); Garrard et al. WO 92/09690 (described, in particular, how the expression of phage); Knappik et al. WO 97/08320 (described library of recombinant human antibodies HuCal); Salfeld et al. WO 97/29131, (describes the preparation of recombinant human antibodies against antigens on human tumor necrosis factor alpha man), and the affinity maturation of recombinant antibodiesin-vitroand Salfeld et al. Provisional patent application U.S. No. 60/126603 and based on it a patent application (in this way describes the preparation of recombinant human antibodies against antigen person (interleukin-12), and the affinity maturation of recombinant antibodiesin-vitro).

Additional described what I screening libraries of recombinant antibodies can be found in scientific publications, such as Fuchs et al. (1991) Bio/Technology 9: 1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et al. (1991) Bio/Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; Barbas et al. (1991) PNAS 88:7978-7982; McCafferty et al. Nature (1990) 348:552-554; Knappik et al. (2000) J. Mol. Biol. 296:57-86.

Alternatively, the use of systems bacteriophobic displays, libraries of recombinant antibodies can Express on the surface of yeast cells or bacterial cells. In WO 99/36569 described methods of obtaining and screening libraries expressed on the surface of yeast cells. In WO 98/49286 more detail the methods of obtaining and screening libraries expressed on the surface of bacterial cells.

In all waysin vitrothe selection process for the enrichment of recombinant antibodies with desirable properties forms an integral part of the process, usually referred to as panning" and often takes the form of affinity chromatography on columns, the bearer of which is attached the structure of the target. Then promising molecule candidates subject to individual determination of their absolute and/or relative affinely, preferably by means of standardized tests dot-blotting, as described above in example 8.

After identifying and quite the characterization of specific antibodies from combinatorial libraries of DNA sequences encoding light and heavy chains of the indicated antibodies, isolated by standard molecular biological methods, for example by PCR amplification of DNA from the packaging of the display (e.g., phage), allocated in the screening library. Nucleotide sequence of the genes for the light and heavy chains of antibodies that can be used to obtain primers for PCR, known to specialists in this field. The set of such sequences described in Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and the database sequences of the germline of the person VBASE.

The antibody or part of an antibody according to the invention can be obtained by means of recombinant gene expression of light and heavy chains of immunoglobulin in the cell host. For recombinant expression of the antibody to the cell-master transferout one or more recombinant expressing vectors carrying DNA fragments encoding the light and heavy immunoglobulin chains of the indicated antibodies, thus expressive light and heavy chains in the cell host and preferably secretion them in an environment in which cultured cells are the host. Antibodies can be isolated from this environment. Standardized methods of recombinant DNA use, h is ordinary to get the genes for the heavy and light chains of the antibody, to insert these genes into recombinant expressing vectors and enter these vectors into cells of the host. Methods of this type are indicated, for example, in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N. Y., (1989), Ausubel, F.M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in the US 4816397 Boss et al.

After obtaining DNA fragments that encode fragments of the VH and VL specific antibodies, these DNA fragments can be further manipulated using standard methods of recombinant DNA, for example, to turn the genes for the variable regions in the genes for full-chain antibodies, genes for Fab fragments or scFv gene. These manipulations include functional binding VL - or VH-encoding DNA fragment with another fragment of DNA encoding another protein, such as a constant region of the antibody or a flexible linker. The term "functionally linked" should be understood in the sense that two fragments of DNA are linked in such a way that the amino acid sequence encoded by the two DNA fragments remain in the frame.

The selected DNA encoding the VH region can be converted to a gene for full-length heavy chains through functional binding DNA that encodes a VH-region, with another DNA molecule that encodes a constant region of the heavy chain (CH1, CH2 and CH3. The gene sequence of the constant region of the heavy chain of human well-known (see, e.g., Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments that overlap the specified area can be obtained by standard PCR amplification. The constant region of the heavy chain may be a constant region from IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE or IgD, where preference is given to the constant region of IgG, particularly IgG1 or IgG4. To obtain the gene for a Fab fragment heavy chain VH-encoding DNA can be functionally linked to another DNA molecule encoding only the constant region CH1 of the heavy chain.

The selected DNA encoding the VL region can be turned into a gene for full-length light chain (and in the gene for the Fab light chain) by a function linking the VL-encoding DNA to another DNA molecule that encodes a constant region CL light chain. The gene sequence of the constant region of the light chain of a man well-known (see Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments that overlap the specified area can be obtained by standard PCR amplification. The constant region of the light chain may be a constant region of Kappa or lambda, depredation give constant region Kappa.

To obtain the gene of scFv VH - and VL-encoding DNA fragments can be functionally linked to another fragment encoding a flexible linker, e.g., amino acid sequence (Gly4-Ser)3so that sequences of VH and VL expressed as a contiguous single-chain protein, where the area of VL and VH are connected to each other through a specified flexible linker (see Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

Single domain VH and VL with affiniscape binding, as described above, can be isolated from libraries of individual domain by the methods described above. Two single chain VH (with or without CH1) or two VL chain or a pair of single chain VH and one VL chain with the desired binding affinity of you can use, as described in the present description, for antibodies according to the invention.

To Express recombinant antibodies or parts of antibodies according to the invention, DNAs encoding partial or full-light and heavy chains, can be embedded in expressing vectors, so that functionally linked genes with the appropriate sequence to control transcription and translation. In this context, the term "functionally linked" should be understood in the sense that the gene antibodies Legerova in the vector so that the sequence counter for the La transcription and translation within the vector serve the function that they regulate transcription and translation of the specified gene antibodies.

It is advisable to choose expressing vector and controls the expression of the sequence so that they were compatible with the cell owner. The gene for the light chain antibody gene and the heavy chain of the antibody can be inserted into separate vectors, or both genes are inserted into the same expressing vector, which is the usual case. Genes antibodies insert into expressing vector by standard methods (e.g., via ligation of complementary restriction sites of cleavage fragment of the antibody gene and the vector, or by ligating blunt ends, unless there are parts of restriction cleavage). Expressing the vector can carry the sequences for the constant regions of the antibodies before inserting sequences for the light and heavy chains. For example, one method is a transformation of sequences of VH and VL genes in a full-sized antibodies by inserting them in expressing vectors already encoding the constant region of the heavy and, accordingly, light chain, therefore, the functional binding fragment of the VH fragment(fragments) of CH in the vector, as well as functional binding fragment LL-CL fragment in the vector.

Additionally or alternatively, recombinant Express the dominant vector can encode a signal peptide, to facilitate the secretion of the chain of the antibody from the host cell. The gene for the specified chain antibodies can be cloned into the vector, thereby connecting the signal peptide in frame with the N-end of the gene for chain antibodies. The signal peptide may be a signal peptide immunoglobulin or a heterologous signal peptide (i.e., signal peptide from a non-immunoglobulin protein). In addition to genes for chain antibodies expressing the vectors according to the invention can contain a regulatory sequence that controls gene expression chain antibodies in a cell-master.

The term "regulatory sequence" is intended to include promoters, enhancers and additional elements to control gene expression (e.g., polyadenylation signals)that control the transcription or translation of genes for chain antibodies. Regulatory sequences of this type are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). The person skilled in the art it is clear that the design expressing vector, including the selection of regulatory sequences may depend on such factors as the subject of transformation of a host cell, the desired power of expression of the protein, etc. of the Preferred regulatory sequences for expression in the cells of the host mlekovita the x include viral elements, providing a strong and constitutive protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the promoter/enhancer CMV), monkey virus 40 (SV40) (such as the promoter/enhancer of SV40), adenovirus (e.g., major late promoter of adenovirus (AdMLP)) and polyoma. Further description of viral regulatory elements, and their sequences, see, for example, in US 5168062 Stinski, US 4510245 Bell et al. and US 4968615 Schaffner et al.

In addition to genes for chain antibodies and regulatory sequences, the recombinant expressing the vectors according to the invention may contain additional sequences, such as sequences that regulate replication of the vector in the cells of the host (for example, the starting point of replication and genes selective markers. Genes selective markers facilitates the selection of host cells, which was introduced in the vector (see, for example, U.S. patent No 4399216, 4634665 and 5179017, all Axel et al.). For example, common genes for selective markers is that they provide the cell-master in which you have entered vector, resistance to cytotoxic drugs, such as G418, hygromycin or methotrexate. Preferred genes are selective markers include the gene for digidrofolatreduktazy (DHFR) (for use in dhfr-the cells of the host with the selection/amplif is used with methotrexate) and the neo gene (for selection with G418).

For the expression of light and heavy chains expressing vector(s)encoding(s) specified heavy and light chains, transferout in a cage-owner by means of standardized methods. Various forms of the term "transfection" are intended to include many ways, accepted for introduction of exogenous DNA into a prokaryotic or eukaryotic cell host, for example, electroporation, calcium-phosphate precipitation, DEAE-dextranase transfection, etc. Although it is theoretically possible to Express the antibodies according to the invention in either prokaryotic or eukaryotic cells-the owners, I prefer the expression of antibodies in eukaryotic cells and, in particular, in the cells of the host mammal, since the probability of correct folding, Assembly and secretion of immunologically active antibodies is higher in such eukaryotic cells, and in particular in mammalian cells than in prokaryotic cells. Published that prokaryotic expression of antibody genes is ineffective to obtain a high yield of active antibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today 6:12-13).

Preferred cells of mammalian hosts for expression of recombinant antibodies according to the invention include CHO cells (including dhfr-the CHO cells, described in Urlaub and Chasin, (198) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with DHFR-selective marker, as described, for example, in R.J. Kaufman and P.A. Sharp (1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and SP2 cells. With the introduction of expressing recombinant vectors encoding antibody genes, in cells of the host mammal, receive antibodies by culturing host cells, while the antibody is not expressed in these cells, the owners, or, preferably, until the antibody is not secreted into the culture medium in which to grow cells-owners. Then the antibodies can be isolated from culture medium using standard methods of protein purification.

Similarly, it is possible to use cell-owners to obtain portions of intact antibodies, such as Fab fragments or scFv molecules. Of course, variants of the above-described method included in the invention. For example, it may be desirable to transliterate cell host DNA encoding either the light chain or the heavy chain (but not both) of the antibodies according to the invention. If there is either light or heavy chain that are not required for binding antigen of interest, then the DNA encoding either such light or the heavy chain, or both, can be removed partially or completely by means of recombinant DNA. The molecules expressed from the same is truncated DNA molecules, likewise included in the antibodies according to the invention. In addition, it is possible to obtain bifunctional antibodies in which the heavy chain and light chain are an antibody of the invention and the other heavy chain and the other light chain are specific for the antigen that is different from the antigen of interest, by means of cross-linking antibodies according to the invention with a second antibody by standard chemical methods.

In a preferred system for recombinant expression of the antibodies according to the invention or its antigennegative part expressing recombinant vector encoding both the heavy chain of the antibody and the light chain of the antibody is introduced into cells dhfr-CHO by transfection mediated by calcium phosphate. In expressing recombinant vector of the genes for the heavy and light chains of the antibodies in each case are functionally linked to regulatory elements CMV enhancer/AdMLP-promoter to ensure a strong transcription of these genes. Recombinant expressing the vector also carries the gene for DHFR, which can be used for selection of dhfr cells-CHO transfected with the vector, using the selection/amplification with methotrexate. Selected transformed cell hosts are cultivated, so expressed thee the white and light chain antibody and the intact antibody isolated from the culture medium. Standardized molecular biological methods used for expressing recombinant vector for transfection of host cells, for selection of transformants, for culturing the above host cells and for obtaining antibodies from the culture medium. Thus, the invention relates to a method for the synthesis of recombinant antibodies according to the invention by culturing a host cell according to the invention in a suitable culture medium until a synthesized recombinant antibody according to the invention. The method also may include the selection of the indicated recombinant antibody from a specific cultural environment.

Alternatively, screening of libraries of recombinant antibodies by phage display technique, other methods known to experts in this field can be used for screening large combinatorial libraries to identify antibodies according to the invention. In principle, you can use any expression system, which reach close physical connection between the nucleic acid and encoded by the antibody and which can be used for screening a suitable nucleic acid sequence based on the properties of the encoded she antibodies.

In one type of alternative system is neither the expression of a library of recombinant antibodies Express in merged form RNA-protein as described in WO 98/31700 Szostak and Roberts, and Roberts, R.W. and Szostak, J.W. (1997) Proc. Natl. Acad. Sci USA 94:12297-12302. In this system, the transmission ofin-vitrosynthetic mRNA bearing on their 3'-end of puromycin, peptidyl-acceptor antibiotic receive covalently fused mRNA and encoded by the peptide or protein. Thus, a specific mRNA in a complex mixture of mRNAs (for example, a combinatorial library), you can concentrate on the basis of the properties of the encoded peptide or protein (e.g. antibody or part thereof), such as the binding of the indicated antibody or specified part thereof with globularia Aβ(20-42) or its derivatives. Nucleic acid sequence that encode antibodies or portions thereof, and which is obtained by screening such libraries, it is possible to Express by means of recombinant methods specified above (for example, in the cells of the host mammal) and can also be subjected to additional maturation affinity either through screening fused mRNA-peptide additional cycles, or the introduction of mutations selected in the source sequence(sequence), or using other methods of affinity maturation of recombinant antibodiesin-vitroin the manner described above.

Combination of methodsin-vivoandin-vitro

Similarly, antibodies according to the invention can be obtained by using from Etania ways in-vivoandin-vitrofor example, the ways in which globular Aβ(20-42) or its derivative at first allow you to work on the repertoire of antibodies in the body is the owner ofin vivoto stimulate the production of antibodies that bind globular Aβ(20-42) or its derivative, and then the additional selection of antibodies and/or maturation of antibodies (i.e. optimization) is carried out using one or more methodsin-vitro. According to one variant of implementation of the combined method of this type may include, first, the immunized non-human animal (e.g. mouse, rat, rabbit, chicken, camel, sheep or goats, or a transgenic variant, or a chimeric mouse) specified globularia Aβ(20-42) or its derivatives to stimulate antibody responses to the antigen and then obtaining and screening of libraries of phage display antibodies using sequences of immunoglobulins lymphocytes stimulatedin vivothrough the impact of the specified globular Aβ(20-42) or derived. The first stage of this combined method can be described above in relation to waysin-vivoby the way, while the second stage of this method can be described above in relation to waysin-vitro. Preferred methods of hyperimmunization not related to chelovekomernyh followed by the screening of libraries of phage display, derived from these stimulated lymphocytes include the methods described BioSite Inc., see, for example, WO 98/47343, WO 91/17271, US 5427908 and US 5580717.

According to another variant implementation of the combined method includes, first, the immunized non-human animal (e.g. mouse, rat, rabbit, chicken, camel, sheep, or goats, or knocked out and/or transgenic variant, or a chimeric mouse) globularia Aβ(20-42) according to the invention or its derivative to stimulate antibody responses to the specified globular Aβ(20-42) or its derivative and selection of lymphocytes producing antibodies having the desired specificity by screening hybridomas (obtained, for example, from immunized animals). Genes for antibodies or single domain antibodies isolated from the selected clones (through standardized ways of cloning, such as polymerase chain reaction with reverse transcriptase) and subjected to affinity maturationin-vitroin order thus to improve the binding properties of the selected antibodies or the selected antibodies. The first stage of this method can be described above in relation to waysin-vivoby the way, while the second stage of this method can be described above in relation to waysin-vitroby the way, in particular, used the eating methods of affinity maturation in-vitrosuch as the methods described in WO 97/29131 and WO 00/56772.

In additional combined method of recombinant antibodies derived from a separate dedicated lymphocytes using a method known to specialists in this field as a way of antibodies from the selected lymphocyte (SLAM) and described in US 5627052, WO 92/02551 and Babcock, J. S. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In this way non-human animal (e.g. mouse, rat, rabbit, chicken, camel, sheep, goat, or transgenic variant, or a chimeric mouse), first, subjected to immunizationin vivoglobularia Aβ(20-42) or its derivatives to stimulate the immune response to the specified oligomer or derivative, and then individual cells secreting specific antibodies are selected using antigen-specific analysis of hemolytic plaques. To this end, globular, or its derivative, or interest of structurally related molecules can be combined with sheep red blood cells using a linker, such as Biotin, thus gaining the ability to identify individual cells secreting antibodies with the appropriate specificity, using analysis of hemolytic plaques. After the identification of cells secreting specific antibodies, cDNA for the variable regions of light and heavy chains derived from cells posredstwom reverse transcriptase, and then these variable regions can be Express in conjunction with the appropriate constant regions of immunoglobulin (e.g., constant regions of a person) in the cells of the host mammal, such as COS cells or CHO. The cells are then-owners, transfetsirovannyh amplificatoare immunoglobulin sequences, derived from selectedin vivolymphocytes, can be subjected to further analysisin-vitroand the selection of thein-vitrothrough the distribution of transfected cells, for example, for selection of cells expressing antibodies with binding affinity of. In addition, immunoglobulin sequences can be manipulatedin vitro.

Antibodies with the required affiniscape defined here, can be selected by holding the dot-blotting, basically as described above. Briefly, the antigen attached to a solid matrix, preferably, put points on the nitrocellulose membrane, in serial dilutions. This is followed by contacting the immobilized antigen of interest antibody followed by detection of the latter by conjugated with the enzyme secondary antibody and colorimetric reaction; when certain concentrations of antibody and antigen, the amount of bound peroxidase antibodies allows determination of affinity. Thus the m the relative affinity of the two different antibodies to the same target or one antibody for two different targets, define here as the ratio of the corresponding quantities associated with the target antibodies observed for the two combinations of antibody-targeted in otherwise identical conditions the dot-blot.

Antibodies that bind to the same epitope as monoclonal antibody 5F7, 10F11, 7C6, 4B7, 6A2, 2F2, 4D10, 7E5, 10C1 or 3B10, you can get essentially known manner.

In the same way described above, any antibody can compete, different patterns of the target is called here "competing" for a certain antibody, if at least one of these structures can specifically reduce the measured binding of the other, preferably by providing overlapping or identical epitope, more preferably, identical epitope.

Competing target molecules can be used for direct selection of antibodies based on their relative affinity for such structures to target. The relative affinity, thus, it is possible to determine directly using competitive analysis, in which distinct forms of competing molecules, for example, differentially labeled competing patterns lead in contact with the antibody of interest and the relative affin the awn antibodies for each of these groups derive from the relative quantities of these molecules, which are associated with the antibody.

This competition can be used for direct enrichment of antibodies having the desired relative affinity to the molecule target, through connection of a target molecule, for which the desirable large affinity to the solid substrate matrix and adding the appropriate amount, preferably a molar excess of competing molecules, for which the desired lower affinity, on Wednesday. Thus, antibodies with the desired relative affiniscape will have a tendency to contact the matrix more strongly than others, and you can get them after washing from less desirable forms, for example, by washing at low salt concentrations and subsequent collection of bound antibodies through its reversible separation from his target with the use of high concentrations of salt. If you prefer, you can perform several cycles of enrichment. In a specific embodiment of the invention, where the genotype underlying the antibodies that are physically associated with this antibody, for example, in the pool hybrid or antigen-exposure phage or yeast cells, you can save the corresponding phenotype.

In another embodiment of the invention using a modified dot-blotting, where the immobilized antigen con is uriroot with the dissolved molecule for binding with the antibody, so the relative affinity of the antibody can be derived from the percentage associated with the immobilized antigen.

Part of the antibodies, such as Fab and F(ab')2fragments can be obtained from whole antibodies using conventional methods, such as cleavage by papain or pepsin. Furthermore, antibodies, part of the antibody and molecules of immunoadhesin can be obtained using standard methods of recombinant DNA.

The present invention also relates to pharmaceutical agents (compositions)containing the antibody according to the invention and, optionally, pharmaceutically suitable carrier. The pharmaceutical compositions according to the invention may also contain at least one additional drug, for example, one or more additional drugs to treat diseases, to facilitate which are applicable antibodies according to the invention. If, for example, the antibody of the invention binds to globularia according to the invention, the pharmaceutical composition may also contain one or more additional medicines for the treatment of disorders in which the activity specified globularia is important.

Pharmaceutically acceptable carriers include any and all solvents, dispersants, coatings, anti-Christ. arterially and antifungal agents, isotonic and slows the absorption of funds, etc. until they are physiologically compatible. Pharmaceutically acceptable carriers include, for example, water, saline, phosphate-saline buffer, glucose, glycerol, ethanol and the like, and combinations thereof. In many cases, preference is given to using isotonic tools, such as sugars, polyalcohol, such as mannitol or sorbitol, or additionally, sodium chloride. Pharmaceutically acceptable carriers may further comprise relatively small amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which increases the half-life or effectiveness of the antibody.

The pharmaceutical composition may be suitable for parenteral administration. Here antibody receive preferably in the form suitable for injection solutions containing antibodies of 0.1-250 mg/ml Suitable for injection solutions can be obtained in liquid or dried form, where the dosage form is in the vessel of flinches or vial, ampoule or a filled syringe. The buffer can contain L-histidine (1-50 mm, preferably 5-10 mm) and have a pH of 5.0-7.0, preferably of 6.0. Additional suitable buffers include, as non-limiting examples, the sodium succinate, the bacilli-citrate, sodium phosphate or potassium phosphate buffers. To regulate toychest solution, you can use sodium chloride at a concentration of 0-300 mm, preferably 150 mm for liquid dosage forms). Cryoprotector, such as sucrose (e.g., 0-10%, preferably, 0.5 to 1.0%), can also be lyophilized dosage form. Other suitable cryoprotectants are trehalose and lactose. Fillers such as mannitol (e.g., 1-10%, preferably 2-4%), can also be lyophilized dosage form. Stabilizers, such as L-methionine (for example, 51-50 mm, preferably 5-10 mm), can be used in both liquid and lyophilized dosage form. Additional suitable fillers are glycine and arginine. You can also use surfactants, such as Polysorbate 80 (e.g., 0-0,05%, preferably of 0.005-0.01%). Additional surfactants are Polysorbate 20 and surfactants BRIJ.

The composition of the invention can have many forms. These include liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., suitable for injection and is suitable for infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. Predpochtitelnei depends on the intended type of injection of therapeutic applications. As a rule, refer to a composition in a form suitable for injection or suitable for infusion solutions, such as compositions similar to the compositions of other antibodies for passive immunization of humans. The preferred method of administration is parenteral (e.g. intravenous, subcutaneous, intraperitoneal or intramuscular). According to a preferred variant implementation of the antibody is administered by intravenous infusion or injection. According to another preferred variant implementation of the antibody is administered by intramuscular or subcutaneous injection.

Therapeutic compositions typically must be sterile and stable under the conditions of production and storage. The composition can be in the form of solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high concentrations of the active substance. Sterile suitable for injection solutions can be obtained through the introduction of active compounds (i.e., antibody) in the required amount in an appropriate solvent, where appropriate, with one or a combination of the above ingredients, as necessary, and then sterilized by filtration of the specified solution.

Dispersion usually get the introduction of the active compound into a sterile medium containing the bulk of the dispersion with the food and when appropriate, other necessary ingredients. In the case of sterile lyophilized powder for receiving suitable for sterile injection solutions drying in a vacuum and spray drying are the preferred methods of preparation, which produce a powder of the active ingredient and, where appropriate, additional desired ingredient from a previously sterile filtered solution. The proper fluidity of the solution can be maintained using, for example, coating, such as a lecithin, by the maintenance, in the case of dispersions, the desired particle size, or through the use of surfactants. In addition, it is possible to achieve a delayed suction suitable for injection of the composition introduced into the composition AIDS in slowing the absorption of, for example, salts of monostearate and gelatin.

Antibodies according to the invention can be activated by a variety of methods known to the person skilled in the art, although the preferred type of introduction for many therapeutic applications is subcutaneous injection, intravenous injection or infusion. The person skilled in the art it is clear that the method and/or the type of administration depends on the desired result. According to specific variants of implementation of the active connection can be obtained with a carrier, which which protects the compound against rapid release, for example, such as compounds with a slow or controlled-release, including implants, transdermal patches and microencapsulation system release. You can use biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyarteritis and polylactic acid. Methods for such compounds is well known to specialists in this area; see, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

According to a specific implementation options, the antibody according to the invention can be administered orally, for example, in an inert diluent or transformed in the course of metabolism edible carrier. The antibody (and optional ingredients, if desired) can also be enclosed in hard or soft gelatin capsule, compressed into tablets, or add directly to food. For oral therapeutic administration antibodies can be mixed with excipients and used in the form of oral tablets, buccal tablets, capsules, elixirs, suspensions, syrups, etc. If you mean to enter the antibody according to the invention by a method other than parenteral may be necessary to select a coating material, warning its inactivation.

This is completed with the invention also relates to a method for inhibiting the activity of globalmenu according to the invention in individuals, suffering from disorders that involved protein β-amyloid, which is especially important activity of these globalmenu according to the invention. This method includes the introduction of at least one antibody according to the invention the individual with the purpose of inhibiting the activity of globularia is associated with the antibody. The specified individual is preferably a human. The antibody according to the invention it is possible to introduce the individual to a person for therapeutic purposes. In addition, the antibody according to the invention it is possible to enter non-human mammals for veterinary purposes or as part of a model of a specific infringement on animals. Such animal models may be applicable for the evaluation of therapeutic efficacy of antibodies of the invention (for example, to test doses and periods of introduction).

Violations, which play a role Globaloney according to the invention, include, in particular, violations that involve the development and/or progression of globularia according to the invention. This, in particular, those violations in which globular according to the invention are expressly or by implication responsible for the pathophysiology of such violations or are a contributor to the development and/or progression of such violations. Accordingly, included those violations is of, in which inhibition of the activity of globalmenu according to the invention can alleviate the symptoms and/or progression of the violation. Such violations may be verified, for example, increased concentrations of globalmenu according to the invention in a body fluid of an individual suffering from specific disorders (e.g., increased concentrations in serum, plasma, CSF, urine etc). It can be used to detect, for example, using antibodies according to the invention. Globaloney according to the invention play an important role in the pathology associated with many disorders are involved in neurodegenerative elements, cognitive disorders, neurotoxic elements and inflammatory elements.

In another aspect of the invention abnormalities that can be treated or prevented include disorders associated with amyloidoses. The term "amyloidosis" refers to a number of disorders characterized by abnormal folding, agglutination, aggregation and/or accumulation of certain proteins (amyloids, fibrous proteins and their predecessors) in various tissues of the body. In Alzheimer's disease and down syndrome damaged nervous tissue, and cerebral amyloid angiopathy (CAA) damaged blood vessels.

The pharmaceutical compositions according to the invention may include a "therapeutically effective amount is or "prophylactically effective amount" of an antibody or part of an antibody according to the invention. "Therapeutically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve the desired therapeutic result. The person skilled in the art can determine therapeutically effective amount of the antibodies or parts of antibodies, and it may vary according to factors such as stage of disease, age, sex and weight of the individual, and the ability of the antibodies or parts of antibodies to cause the desired response in the individual. Therapeutically effective amount is the amount at which therapeutically beneficial effects of antibodies or parts of antibodies is superior to any toxic or adverse effects. "Prophylactically effective amount" refers to an amount effective at dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, since a prophylactic dose is used for the subjects to disease or at an earlier stage of disease, the prophylactically effective amount will be less than therapeutically effective amount.

In addition, the present invention relates to an additional method of prevention or treatment of Alzheimer's disease in a patient in need of such prevention or treatment. This with the ESP includes a stage of introduction of the patient mentioned above vaccines in number, sufficient to provide for the prevention or treatment.

In addition, the present invention relates to a method of identifying compounds suitable for active immunization of the patient, for which predicted the development of amyloidosis, such as Alzheimer's disease. This method includes: 1) exposure of one or more compounds of interest to the influence of one or more of the antibodies described above, in the course of time and under conditions suitable for binding of one or more compounds with the antibody or antibodies; 2) identification of the compounds by contacting the antibody or antibodies, where the identified compounds to be used for active immunization of the patient, for which predicted the development of amyloidosis, such as Alzheimer's disease.

In the framework of the diagnostic use of antibodies qualitative or quantitative determination of specific globular serves, in particular, for the diagnosis-related disease forms of β-amyloid. In this context, specificity refers to the capability of detection of specific globular, or its derivative, or mixtures thereof, with sufficient sensitivity. Antibodies according to the invention mainly have a concentration threshold of detection of less than 10 ng/ml of sample, preferably less than 1 ng/ml sample, and especially before occhialino, less than 100 PG/ml of sample, which means that at least the concentration of globular per ml of sample specified in each case, primarily, lower concentrations can be detected by antibodies according to the invention.

Detection was performed immunological. It is, in principle, can be performed using any analytical or diagnostic method of analysis, which use antibodies, including methods, agglutination and precipitation, immunoassays, immunohistochemical methods and means of Western blot turns, for example, Western blotting or, preferably, methods of the dot-blotting. This includes howin vivofor example, the visualization techniques.

The use of immunoassays is predominant. Suitable as a competitive immunoassays, i.e. the test, where the antigen and labeled antigen (indicator) compete for binding to the antibody, and sandwich immunoassays, i.e. the test, where the binding of specific antibodies to the antigen detected by a second, usually labeled antibodies. These tests can be either homogeneous, i.e. without separation of solid and liquid phase, or heterogeneous, i.e. the associated label is separated from the unbound label, for example, by means associated with the solid phase antibody. Depending on the labeling and method of measurement of various courtesans the gene and homogeneous immunoassay formats can be classified into specific classes, for example, RIA (radioimmunoassay analyses), ELISA (enzyme linked immunosorbent assay), FIA (fluorescent immunoassay), LIA (fluorescent immunoassay), TRFIA (FIA with a temporal resolution), IMAC (immunoactivity), EMIT (enzyme-multiplicity immune test), TIA (turbidimetric immunoassay), I-PCR (immuno-PCR).

For the quantitative determination of globular according to the invention preference is given to competitive immunoassays, in which a certain number of labeled derivative globularia that serves as an indicator that competes with globularia from a sample containing an unknown amount of unlabeled globalmenu)subject to quantification, for binding with the antibody. The amount of antigen, i.e. the number of globular in the sample can be determined by the number of overset indicator using the standard curve.

It is shown that of the marks available for these purposes, the enzymes are preferred. You can use, for example, systems based on peroxidases, such as horseradish peroxidase, alkaline phosphatase and β-D-galactosidase. Specific substrates, the transformation of which it is possible to monitor, for example photometrically available for these enzymes. Suitable substrate system based on p-nitrophenylphosphate (p-NPP), 5-bromo-4-chloro-3-indolylmethane/nitrocine tet is Azalee (BCIP/NPT), solid red/naphthol-AS-TS phosphate for alkaline phosphatase; 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPT), 3,3',5,5'-tetramethylbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid, 3-dimethylaminobenzoyl acid (DMAB) and 3-methyl-2-benzothiazolinone (MBTH) for peroxidase; o-nitrophenyl-β-D-galactoside (o-NPG), p-nitrophenyl-β-D-galactoside and 4-methylumbelliferyl-β-D-galactoside (MUG) for β-D-galactosidase. In many cases, these substrate systems are commercially available in ready to use form, for example in the form of pills, which can also contain additional reagents, such as suitable buffers, etc..

The indicators used can be a labeled Globaloney. In this sense, the specific globular can be determined by labeling subject to definition globular and use it as an indicator.

Attach labels to globularia for the indicators can be accomplished by way of the known substance. The comments above regarding derivatization of globalmenu according to the invention relate to the analogy. In addition, there are a number of labels, appropriately modified for conjugation with proteins, for example, Biotin-, avidin-, extrawide - or streptavidin-conjugated enzymes, maleinimide-activated fer the coefficients etc. To obtain the indicator can be the reaction of these labels directly from the oligomer or, globularia. If, for example, use conjugate streptavidin-peroxidase, then this is, firstly, requires biotinidase of globularia. It is used, respectively, in the reverse order. Suitable for this purpose, methods are also known specialist in this field.

If the selected format heterogeneous immunoassay, the complex antigen-antibody can be separated by its binding to the substrate, for example through antiidiotypic antibody attached to a given substrate, for example, an antibody directed against rabbit IgG. Suitable substrate, in particular a titration microplate coated with appropriate antibodies, known and partially commercially available.

In addition, the present invention relates to kits for immunoassays containing at least one antibody as described above, and additional components. These sets are usually represented in the form of a packaged unit, the combination of means for assessing globular according to the invention. For the purposes of so simple manipulations, as far as possible, these means preferably provide in ready to use form. In the preferred arrangement, the immunoassay offer in the form of a set. Nab the R typically contains a variety of containers for separate layout components. All components can be provided in ready-to-use solution, in the form of a concentrate for dilution or dry matter or lyophilisate for solution or suspension; some or all of the components can be refrigerated or stored at room temperature until use. The serum is preferably frozen, for example at -20°C, so in these cases, components for immunoassay before use must be stored, preferably at temperatures below freezing.

Additional components supplied with components for immunoassays depend on the type of immunoassay. Usually the standard protein, an indicator that may or may not be necessary, and the control serum supplied with anticorodal. In addition, you can also include microplates for titration, preferably, coated with an antibody, buffers, for example, for testing, for washing or for the conversion of the substrate, and the substrate for the enzyme.

General principles of immunoassays, as well as the preparation and use of antibodies as an aid in the laboratory and the hospital can be found, for example, in Antibodies, A Laboratory Manual (Harlow, E. and Lane, D., Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1988).

Thus, the present invention is also relative to the Xia to the method for the diagnosis of amyloidosis, for example, Alzheimer's disease, the patient, presumably suffering from this disease. This method includes the steps: 1) highlight a biological sample from the patient; 2) contacting a biological sample with at least one of the above antibodies for a time and under conditions suitable for formation of complexes of antigen/antibody; and 3) detecting the presence of complexes of antigen/antibody in the specified sample, where the presence of complexes indicates the diagnosis of amyloidosis, such as Alzheimer's disease, in a patient. The antigen may represent, for example, globular or part thereof, or a fragment having the same functional properties as the full sized globular (e.g., binding activity).

In addition, the present invention relates to another method for the diagnosis of amyloidosis, such as Alzheimer's disease, the patient, presumably suffering from this disease. This method includes the steps: 1) highlight a biological sample from the patient; 2) contacting a biological sample with an antigen for a time and under conditions suitable for formation of complexes of antibody/antigen; 3) adding a conjugate to the resulting complexes of antibody/antigen for a period of time and under conditions suitable to allow the conjugate swastic is linked antibody where the conjugate contains one of the above-described antibodies attached to making the signal connection, capable of producing measurable detection signal; 4) detection of the presence of antibodies which may be present in the biological sample by detecting the signal produced by producing a signal connection, where the signal indicates the diagnosis of amyloidosis, such as Alzheimer's disease, in a patient. The antigen may be globular or part thereof or a fragment having the same functional properties as the full sized globular (e.g., binding activity).

The present invention relates to an additional method for the diagnosis of amyloidosis, such as Alzheimer's disease, the patient, presumably suffering from amyloidosis, such as Alzheimer's disease. This method includes the steps: 1) highlight a biological sample from the patient; 2) contacting a biological sample with anti-antibody, where the anti-antibody is specific for one of the above antibodies for a time and under conditions suitable to allow formation of complexes of anti-antibody/antibody complexes where contain the antibody present in the biological sample; 2) adding a conjugate to the resulting complexes of anti-antibody/antibody lane on the od of time and under conditions suitable to allow the conjugate to contact with the associated antibody, where the conjugate contains an antigen that is associated with producing a signal connection, capable of producing measurable detection signal; 3) detection of the signal produced by producing a signal connection, where the signal indicates the diagnosis of amyloidosis, such as Alzheimer's disease, in a patient.

The present invention relates also to a kit, comprising: a) at least one of the above-described antibody and b) a conjugate containing an antibody attached to making the signal connection, where the antibody of the conjugate is different from the selected antibodies.

The present invention relates also to a kit, comprising: a) an anti-antibody against one of the above-described antibody and b) a conjugate containing the antigen attached to making the signal connection. The antigen may be globular or its fragment or portion, having the same functional characteristics as the full-sized globular (e.g., binding activity).

In one of the diagnostic embodiments of the present invention the antibody of the present invention or part thereof to cover the solid phase (or it is present in the liquid phase). Then test or biological sample (e.g., whole blood, cerebrospinal liquid is, serum etc) then lead into contact with the solid phase. If the pattern is present antigen (for example, globular), such antigens are associated with antibodies on the solid phase and then detects either directly or indirectly. The direct method involves a simple detection of the actual complex and, thus, the presence of antigens. Indirect way, to the related add antigen conjugate. The conjugate contains a second antibody that binds with an associated antigen attached to making the signal connection or label. If the second antibody binds with an associated antigen producing a signal connection produces a measurable signal. Then, this signal indicates the presence of antigen in the test sample.

Examples of solid phases used for diagnostic immunoassays, are porous and non-porous materials, latex particles, magnetic particles, microparticles (see U.S. patent No. 5705330), beads, membranes, microtiter wells for micrometrology and plastic tubes. The choice of material of the solid phase and method of labelling an antigen or antibody present in the conjugate, if desirable, is determined on the basis of the performance characteristics desired format of analysis.

As indicated above, the conjugate (or reagent indicator) contains an antibody (or, for the, anti-antibody, depending on the analysis), attached to making the signal connection or label. This produces a signal connection or "tag" can be used by itself or it may react with one or more additional compounds to obtain quantifiable detection of the product. Examples of producing the signal connections include Chromogens, radioactive isotopes (e.g., 125I, 131I, 32P, 3H, 35S and 14C), chemiluminescent compounds (e.g., acridine), particles (visible or fluorescent), nucleic acids, complexing funds or catalysts such as enzymes (e.g. alkaline phosphatase, acid phosphatase, horseradish peroxidase, beta-galactosidase and ribonuclease). In the case of an enzyme (e.g. alkaline phosphatase or horseradish peroxidase), adding chrome, fluoro or luvhengo substrate results in measurable detection signal. Other systems of detection, such as fluorescence with time resolution, fluorescence internal reflection, amplification (such as polymerase chain reaction) and Raman spectroscopy, are also applicable.

Examples of biological fluids that can be tested by the above-described immunoassays include plasma, whole blood, dried whole blood, savor the weave, cerebrospinal fluid, or aqueous or organic-aqueous extracts of tissues and cells.

The present invention relates also to a method of detecting the presence of antibodies in the tested sample. This method includes the stages of: (a) contacting the test sample suspected of containing the antibodies with anti-antibody specific for the antibody in the sample from the patient, over a period of time and under conditions suitable to allow formation of complexes of anti-antibody/antibody, where the anti-antibody is an antibody of the present invention, which binds to the antibody in the sample from the patient; (b) adding a conjugate to the resulting complexes of anti-antibody/antibody, where the conjugate contains antigen (binding to an anti-antibody)attached to making the signal connection is capable of producing measurable detection signal; (d) detecting the presence of antibodies that may be present in the test sample by detecting the signal produced by producing a signal connection. You can use the control or calibrator containing antibody against anti-antibodies.

The sets also included in the scope of the present invention. More specifically, the present invention relates to kits for determining the presence of antigens (for example, globularia) the patient is a, presumably suffering from Alzheimer's disease or other condition characterized by cognitive impairment. In particular, a kit for determining the presence of antigens in the test sample contains: a) an antibody, as defined here, or part thereof; (b) a conjugate containing the second antibody with specificity to the antigen), attached to making the signal connection, capable of producing measurable detection signal. The kit may also contain a control or calibrator containing the reagent to bind to the antigen.

The present invention relates also to a kit for detection of antibodies in the tested sample. The kit may contain: a) an anti-antibody (for example, one of rassmatrivaemogo of the invention)specific for specific antibodies, (b) an antigen or portion thereof, as defined above. You can also include a control or calibrator containing the reagent to bind to the antigen. More specifically, the kit may contain: a) an anti-antibody (such as one of the present invention)specific antibodies (b) a conjugate containing the antigen (for example, globular)attached to making the signal connection, capable of producing measurable detection signal. And in this case, the kit may also contain a control or calibrator containing reagent that communicates with the Antiga is om.

The kit may also contain one container such as a vial, bottle or strip, each container with the prepared solid phase, and other containers containing the corresponding conjugates. These kits can also contain bottles or containers other reagents required for analysis, such as reagents for washing, processing and reagents, indicators.

It should also be noted that the invention relates not only to the full-size antibodies, described above, but also to their parts or fragments, for example, part of the Fab. Moreover the present invention relates to any antibody having the same properties as these antibodies, for example, from the point of view of binding specificity, structure, etc.

The advantages according to the invention is:

Through immunization globularia Aβ(20-42) you can get different monoclonal antibodies that differ in their tolerance or recognition for various oligomers Aβ(1-42) oligomers Aβ(X-42), as determined by competitive dot-blotting as described above. This allows the development of antibodies directed against the truncated N-Termini of Aβ oligomers with an optimal ratio between the effect of improving cognitively desired specificity for other forms of Aβ and minimal side profile the effects. Suddenly, Aβ(1-42) and globular Aβ(12-42), despite the content of the structural element, are only partially recognizable by antibodies produced using advanced truncated globular Aβ(20-42) as antigen.

By restricting specific oligomeric forms of Aβ based on the structural principle (i.e. use truncated at the N end of globalmenu Aβ), received the antibody profile, active immunization which is highly specific for oligomeric forms of Aβ. The same remains in force for monoclonal antibodies for use in passive immunization. The advantage of this specific strategy of immunization (active and passive) is that it will not cause an immune response against Aβ monomers and Aβ peptides in the States of fibrillar aggregation or sAPPα. This provides an advantage in several ways.

1) In the form of insoluble Aβ plaques Aβ peptides in the States of fibrillar aggregation constitute the main part of the full pool of Aβ peptides in the brain in AD. Mass release of Aβ by dissolving plaques Aβ-induced reaction of antibodies against Aβ with these plaques should be considered as adverse. Then this mass release of Aβ will cross the blood-brain barrier to enter the bloodstream and potentially increase R is IC microchromosome. In addition, the test ELAN the same strategy immunization with fibrillar forms of Aβ peptide demanded the termination of the experiment due to 6% of cases debut meningoencephalitis.

2) Immune responses directed against Monomeric forms of Aβ peptides, are undesirable, as it was possible to show that later they can influence the effects of improving cognitively.

3) Immune responses directed against sAPPα, in the same way are undesirable because they can lead to a reaction against physiologically existing protein-precursor APP and, thus, to autoimmune reactions. Moreover, it is also shown that sAPPα may also influence the effects of improving cognitively.

4) Response directed against vascular Aβ peptide in the form of CAA, also should be discontinued to avoid unwanted side-effect of microchromosome (antibodies against the Central part of Aβ, which, moreover, is not associated with Aβ peptides, aggregated in the form of CAA, induce less microchromosome compared with antibodies against the N-Terminus, see above).

5) Antibodies specifically reactive with Aβ oligomers have a higher bioavailability compared to pathophysiologically relevant species of Aβ, as they will not be contacted, for example, with fibrillar Aβ and thus becomes n is available for therapeutic effect.

Information escrow: hybridoma producing monoclonal antibody 5F7, deposited in the American type culture collection, 10801 University Boulevard, Manassas, Virginia 20110, 01 December 2005 within the framework of the Budapest Treaty and received designation PTA-7241. In addition, hybridoma producing monoclonal antibody 10F11, deposited in the American type culture collection, 10801 University Boulevard, Manassas, Virginia 10801, 01 December 2005 within the framework of the Budapest Treaty and received designation PTA-7239. In addition, hybridoma producing monoclonal antibody 4B7, deposited in the American type culture collection, 10801 University Boulevard, Manassas, Virginia 10801 01 December 2005 within the framework of the Budapest Treaty and received designation PTA-7242, and hybridoma producing monoclonal antibody 7C6, deposited in the American type culture collection, 10801 University Boulevard, Manassas, Virginia 10801, 01 December 2005 within the framework of the Budapest Treaty and received designation PTA-7240. In addition, hybridoma producing monoclonal antibody 6A2, deposited in the American type culture collection, 10801 University Boulevard, Manassas, Virginia 10801, February 28, 2006, within the framework of the Budapest Treaty and received designation PTA-7409, and hybridoma producing monoclonal antibody 2F2, deposited in the American type culture collection, 10801 University Boulevard, Manassas, Virginia 10801, February 28, 2006, in the framework of the Budapest Treaty and received designation PTA-7408. Hybridoma producing monoclonal antibody 4D10, deposited in the American type culture collection, 10801 University Boulevard, Manassas, Virginia 10801, February 28, 2006, within the framework of the Budapest Treaty and received designation PTA-7405. Hybridoma producing monoclonal antibody 7E5, deposited in the American type culture collection, 10801 University Boulevard, Manassas, Virginia 10801, 16 August 2006 within the framework of the Budapest Treaty and received designation PTA-7809. Hybridoma producing monoclonal antibody 10C1, deposited in the American type culture collection, 10801 University Boulevard, Manassas, Virginia 10801, 16 August 2006 within the framework of the Budapest Treaty and received designation PTA-7810. Hybridoma producing monoclonal antibody 3B10, deposited in the American type culture collection, 10801 University Boulevard, Manassas, Virginia 10801, 01 September 2006, within the framework of the Budapest Treaty and received designation PTA-7851. All deposits made on behalf of Abbott Laboratories, 100 Abbott Park Road, Abbott Park, Illinois 60064 (US).

Drawings

The figure 1 shows the exclusion chromatogram Aβ(1-42) and Aβ(1-40). The monomer Aβ(1-42) was dissolved in (A) 0.1% of NH4OH, B) 70% formic acid C) of 0.1% NaOH and D) Aβ(1-40) was dissolved in 0.1% NaOH. Then the sample was further diluted 1:10 in 20 mm NaH2PO4, 140 mm NaCl, pH 7,4. These samples were incubated for 5 min (left column) or 1 hour (right column) after rastvorenii ambient temperature, then was applied on the column for exclusion chromatography;

In figure 2 (A) shows the SDS PAGE of standard proteins (protein molecular weight marker, lane 1); preparation of fibrils of Aβ(1-42); control (lane 2); preparation of fibrils of Aβ(1-42) + mAb 5F7, 20 h, 37°C, supernatant (lane 3); preparation of fibrils of Aβ(1-42) + mAb 5F7, 20 h, 37°C, the precipitate (lane 4); preparation of fibrils of Aβ(1-42) + mAb 6E10, 20 h, 37°C, supernatant (lane 5); preparation of fibrils of Aβ(1-42) + mAb 6E10, 20 h, 37°C, the precipitate (lane 6);

(B) shows the results of quantitative analysis of mAb associated with Aβ fibrils in percent of total antibodies;

The figure 3 presents the histogram, which shows the results of the test object identification transgenic APP mice/L after active immunization with Aβ monomers(1-42) in 0.1% NH4OH, globularly Aβ(1-42) and globularly Aβ(20-42) compared with wild-type mice (positive control) and mice APP/L, treated with PBS (negative control), where the circles shown significant differences with PBS treated mice APP/L, and the stars shown highly significant differences with random level (50%) according to the secondary t-test after P<0,05 in ANOVA for differences between groups;

The figure 4 shows the dot-blots for reactivity 100 pmol/µl (range A); 10 pmol/µl (range B); 1 pmol/µl (range C); 0.1 pmol/µl (range D) and 0.01 pmol/μl (range E) globular Aβ(1-42) (column 1); pre-processing the aqueous HFIP monomer Aβ(1-42) in Pluronic F68 (column 2); globular Aβ(20-42) (column 3); globular Aβ(12-42) (column 4); HFIP pretreated monomer Aβ(1-40) in DMSO (column 5); the monomer Aβ(1-42), NH4OH (column 6); preparation of fibrils of Aβ(1-42) (column 7); and sAPPα from Sigma (column 8) with different antisera obtained after active immunization of mice APP/L Tg-globularia Aβ(20-42);

Figure 5 shows a histogram, which shows the concentration of soluble and insoluble Aβ(1-42) and Aβ(1-40) peptide in extracts of brain of APP/PS1 Tg mice actively immunized with either the monomer Aβ(1-42) (0.1% of NH4OH), or globularia Aβ(1-42)or globularia Aβ(20-42)or medium as control;

The figure 6 shows a histogram that shows the results of the test object identification transgenic APP mice/L after passive immunization with antibodies 5F7, 10F11 and 7C6 against globular Aβ(20-42) compared to control mice for A) each antibody separately and (B) for all antibodies, taken together;

In figure 7 (A) shows the analysis of dot-blotting specificity of different antibodies against Aβ (-6E10, -5F7, -4B7, -10F11, -6A2, -4D10, -3B10, -2F2, -7C6, -7E5, -10C1). Monoclonal antibodies tested here, obtained by active immunization of mice with globularia Aβ(20-42) with subsequent selection of merged cells hybridoma (except commercially available 6E10, Signet No 9320). Selected forms of Aβ were applied in serial dilutions and what was kopirovali with the appropriate monoclonal antibodies for the immune response:

1) the monomer Aβ(1-42), 0.1 % of NH4OH;

2) the monomer Aβ(1-40), with 0.1% NH4OH;

3) the monomer Aβ(1-42), a 0.1% NaOH;

4) the monomer Aβ(1-40), with 0.1% NaOH;

5) globular Aβ(1 -42);

6) globular Aβ(12-42);

7) globular Aβ(20-42);

8) preparation of fibrils of Aβ(1-42);

9) sAPPα (Sigma) (first point: 1 pmol).

(B) quantification was performed using a densitometric analysis of the intensity. For each form of Aβ were evaluated by only one point corresponding to only the lowest concentration of antigen, provided that she had a relative density of greater than 20% of the relative density of the latter is uniquely determined point of globular Aβ(20-42) (threshold). The threshold value was determined for each dot-blot independently. The value indicates the ratio between the recognition of globular Aβ(20-42) and the corresponding form of Aβ for this antibody;

The figure 8 shows the binding of the antibody at various concentrations with the cross-sections of the neocortex of patients with Alzheimer's disease (AD) or old APP transgenic mice:

A) Verification of amyloid deposits by Congo red staining in the form of plaques in the brain tissue and in the form of cerebral amyloid angiopathy (CAA) in the brain of transgenic mouse APP line Tg2576 and patients with AD (RZ55);

B) Strong staining parenchymal deposits of Aβ (amyloid plaques) in patients with AD (RZ16) presets is there only with 6G1 and with the commercially available antibody 6E10 (left column), while for antibodies 5F7, 2F2 and 6A2 (second column), 4D10, 10F11 and 3B10 (third column) and 7C6, 7E5 and 10C1 (right column) showed no staining. All antibodies were used at a concentration of 0.7 μg/ml;

C) Strong staining parenchymal deposits of Aβ (amyloid plaques) in Tg2576 at the age of 19 months was present only with 6G1 and with the commercially available antibody 6E10 (left column), while for antibodies 5F7, 2F2, and 6A2 (second column), 4D10, 10F11 and 3B10 (second column) and 7C6, 7E5, 10C1 and (right column) showed no staining. All antibodies were used at a concentration of 0.7 μg/ml;

D)-(G) Quantitative analysis of staining of Aβ plaques on histological images using image analysis. The values of optical density (0% = no staining) was calculated values for plaques in grayscale, which read values for the background tissue grayscale: D) Staining of 0.7 µg/ml antibody for old Tg2576 mice, E) staining of 3 different concentrations of antibodies to mice APP/L, F) staining of 0.7 mg/ml of antibody for a patient with AD (RZ55), (G) staining of 3 different concentrations of antibodies for a patient with AD (RZ16). The difference between painting the commercially available antibody 6E10 (asterisks) and 4G8 (circles) and all other antibodies (three stars/circle: p<0,001 compared with control; secondary t-test, Bonferroni after ANOVA with p<0,001) were evaluated statistically (D1F). In (E) and (G) for all antibodies, except 6G1 always showed significantly less staining than for the commercially available antibody 6E10 and 4G8 (p<0,001 in the secondary t-test after p<0.001 in ANOVA). H) Strong staining of Aβ deposits in blood vessels (arrows) is present only with 6G1 and the commercially available antibody 6E10 (left column), while for antibodies 5F7, 2F2 and 6A2 (second column), 4D10, 10F11 and 3B10 (third column) and 7C6, 7E5, 10C1 and (right column) showed no staining. All antibodies were used at a concentration of 0.7 µg/ml Quantitatively similar situation was found for mice Tg2576 (not shown here);

Figure 9. The titer of antibodies against Aβ and the profile of selectivity in dot-blotting of plasma TG2576 mice after approximately one year after active immunization. In plasma samples of mice Tg2576 after approximately one year after the last immunization a) globularia Aβ (20-42), B) globularia Aβ (12-42), (C) a monomer Aβ (1-42) and (D) a carrier, through the dot-blot was estimated that antibodies against Aβ produced and present.

1) globular Aβ (1-42);

2) the monomer Aβ (1-42), HFIP pretreated in 0.1% Pluronic F68;

3) globular Aβ (20-42);

4) globular Aβ (12-42);

5) the monomer Aβ (1-40), HFIP pretreated with 5 mm in DMSO;

6) the monomer Aβ (1-42), 0.1% of NH4OH;

7) preparation of fibrils of Aβ (1-42);

8) sAPPα (Sigma); (the first point: 1 pmol).

On Figo is e 10 shows a table where summative levels globular Aβ(20-42) in the brain tissue of people with Alzheimer's disease, and controls without dementia;

The figure 11 shows the nucleotide and amino acid sequences of variable heavy and light chains of monoclonal antibodies (mAb) as follows (complementarity determining region (CDRs) are underlined in each amino acid sequence):

33
11SEQ ID NO:Sequence typeChainmAb
A11nucleotidevariable heavy (VH)5F7
A22nucleotidevariable light (VL)5F7
A13amino acidvariable heavy (VH)5F7
A24amino acidvariable l is HCA (VL) 5F7
B15nucleotidevariable heavy (VH)10F11
B26nucleotidevariable light (VL)10F11
B17amino acidvariable heavy (VH)10F11
B28amino acidvariable light (VL)10F11
C19nucleotidevariable heavy (VH)7C6
C210nucleotidevariable light (VL)7C6
C111amino acidvariable heavy (VH) 7C6
C212amino acidvariable light (VL)7C6
D113nucleotidevariable heavy (VH)4B7
D214nucleotidevariable light (VL)4B7
D115amino acidvariable heavy (VH)4B7
D216amino acidvariable light (VL)4B7
E117nucleotidevariable heavy (VH)2F2
E218nucleotidevariable light (VL)2F2
E119amino acidvariable heavy (VH)2F2
E220amino acidvariable light (VL)2F2
F121nucleotidevariable heavy (VH)6A2
F222nucleotidevariable light (VL)6A2
F123amino acidvariable heavy (VH)6A2
F224amino acidvariable light (VL)6A2
G125nucleotidevariable heavy (VH)4D10
G226nucleotidevariable light (VL)4D10
G127amino acidvariable heavy (VH)4D10
G228amino acidvariable light (VL)4D10
H129nucleotidevariable heavy (VH)7E5
H230nucleotidevariable light (VL)7E5
H131amino acidvariable heavy (VH)7E5
H232amino acidvariable light (VL)7E5
I1nucleotidevariable heavy (VH)10C1
I234nucleotidevariable light (VL)10C1
I135amino acidvariable heavy (VH)10C1
I236amino acidvariable light (VL)10C1
J137nucleotidevariable heavy (VH)3B10
J138amino acidvariable heavy (VH)3B10

The following examples are intended to illustrate the invention without limiting its scope.

Example 1: Getting globalmenu

a) globular Aβ(1-42)

Synthetic peptide Aβ(1-42) (H-1368, Bachem, Bubendorf, Switzerland) suspended in 100% 1,,1,3,3,3-hexamer-2-propanol (HFIP) at 6 mg/ml and incubated for complete dissolution shaking at 37°C for 1.5 hours HFIP acts as a destroyer of hydrogen bonds, and it is used to remove pre-existing structural inhomogeneity in the Aβ peptide. HFIP was removed by evaporation in a Speed Vac and Aβ(1-42) resuspendable to a concentration of 5 mm in dimethyl sulfoxide and treated with ultrasound for 20 sec. HFIP pretreated Aβ(1-42) was dissolved in phosphate-buffered saline (PBS) (20 mm NaH2PO4, 140 mm NaCl, pH 7,4), 400 μm was added 1/10 volume of 2% sodium dodecyl sulfate (SDS) (H2O) (final concentration of 0.2% SDS). Incubation for 6 h at 37°C was obtained intermediate globular Aβ(1-42) 16/20-kDa (short form globular oligomer). Globular Aβ(1-42) 38/48-kDa received additional dilution with three volumes of H2O and incubation for 18 h at 37°C. After centrifugation at 3000g for 20 min the sample was concentrated by ultrafiltration (cut-off of 30 kDa), were dialyzed against 5 mm NaH2PO4, 35 mm NaCl, pH of 7.4, centrifuged at 10,000 g for 10 min and supernatant was collected, containing the 38/48-kDa globular Aβ(1-42). As an alternative to dialysis, 38/48-kDa globular Aβ(1-42) can also precipitate a ninefold excess (about./about.) ice-cold solution of methanol/acetic acid (33% methanol, 4% acetic acid) for 1 h at 4°C. Then 38/48-kDa globular Aβ(1-42) besieged (10 min at 16200g), resuspendable 5 mm NaH2PO4, 35 mm NaCl, pH7,4 and brought the pH to 7.4.

b) Cross-stitched globular Aβ(1-42)

Synthetic peptide Aβ(1-42) (H-1368, Bachem, Bubendorf, Switzerland) suspended in 100% 1,1,1,3,3,3-hexamer-2-propanol (HFIP) at 6 mg/ml and incubated for complete dissolution shaking at 37°C for 1.5 hours HFIP acts as a destroyer of hydrogen bonds, and it is used to remove pre-existing structural inhomogeneity in the Aβ peptide. HFIP was removed by evaporation in a SpeedVac and Aβ(1-42) resuspendable to a concentration of 5 mm in dimethyl sulfoxide and treated with ultrasound for 20 sec. HFIP pretreated Aβ(1-42) was dissolved in phosphate-buffered saline (PBS) (20 mm NaH2PO4, 140 mm NaCl, pH 7,4), 400 μm was added 1/10 volume of 2% sodium dodecyl sulfate (SDS) (H2O) (final concentration of 0.2% SDS). Incubation for 6 h at 37°C was obtained intermediate globular 16/20-kDa Aβ(1-42) (short form globular oligomer). 38/48-kDa globular Aβ(1-42) received additional dilution with three volumes of H2O and incubation for 18 h at 37°C. Now conducted cross-stitching 38/48-kDa of globular Aβ(1-42) by incubation with 1 mm glutaraldehyde for 2 h at room temperature (RT) 21°C followed by treatment with ethanolamine (5 mm) for 30 min at RT.

c) globular Aβ(20-42)

1,59 ml of globular Aβ(1-42), obtained according to example 1a was mixed with 38 ml of buffer (50 mm MES/NaOH, pH 4) and 200 μl of a solution thermolysin 1 mg/ml (Roche) in water. The reaction mixture was stirred at RT for 20 h and Then was added 80 μl of 100 mm EDTA, pH 7,4, in water and, in addition, brought the SDS content in the mixture to 0.01% with 400 μl of SDS solution concentration of 1%. The reaction mixture was concentrated to approximately 1 ml by Centriprep tube 15 ml 30 kDa. The concentrate was mixed with 9 ml of buffer (50 mm MES/NaOH, 0,02% SDS, pH 7,4) and again concentrated to 1 ml of the Concentrate were dialyzed at 6°C against 1 l of buffer (5 mm sodium phosphate, 35 mm NaCl) in a dialysis tube for 16 hours Then drove the content of SDS in dialysate to 0.1% using SDS solution in water with a concentration of 2%. The sample was centrifuged at 10000g for 10 min, and supernatant was collected of globular Aβ(20-42).

d) globular Aβ(12-42)

2 ml of globular Aβ(1-42), obtained according to example 1a was mixed with 38 ml of buffer (5 mm sodium phosphate, 35 mm sodium chloride, pH of 7.4) and 150 μl of 1 mg/ml endoprotease GluC (Roche) in water. The reaction mixture was stirred for 6 h at RT and then added an additional 150 μl of 1 mg/ml endoprotease GluC (Roche) in water. The reaction mixture was stirred at RT for 16 h, followed by addition of 8 μl of 5 M solution DIFP. The reaction mixture was concentrated to approximately 1 ml by Centriprep tube 15 ml 30 kDa Centriprep. The concentrate was mixed with 9 ml of buffer (5 mm sodium phosphate, 35 mm sodium chloride, pH 7,4) and again concentrated to 1 ml Conc Strat were dialyzed at 6°C against 1 l of buffer (5 mm sodium phosphate, 35 mm NaCl) in a dialysis tube for 16 hours Then drove the content of SDS to 0.1% using SDS solution in water with a concentration of 1%. The sample was centrifuged at 10000g for 10 min and supernatant was collected of globular Aβ(12-42).

Example 2: size-Exclusion chromatography of various drugs monomer Aβ(1-42) monomer Aβ(1-40)

Aβ(1-42), 0.1% of NH4OH:

1 mg of Aβ(1-42) (Bachem, catalog # H-1368) were dissolved in 500 μl of 0.1% NH4OH in H2O and stirred for 1 min. at ambient temperature. The sample was centrifuged for 5 min at 10000g. Collected supernatant. The concentration of Aβ(1-42) in the supernatant was determined according to the method of Bradford (BIO-RAD).

The sample for 5 min:

20 μl of the supernatant containing Aβ(1-42) in 0.1% NH4OH, bred 20 mm NaH2PO4, 140 mm NaCl, pH 7.4 to a concentration of Aβ(1-42) 0.2 mg/ml of Sample were incubated for 5 min at ambient temperature. Then 100 μl were analyzed exclusion chromatography (SEC).

The sample for 1 hour:

20 μl of the supernatant containing Aβ(1-42) in 0.1% NH4OH, was dissolved in 20 mm NaH2PO4, 140 mm NaCl, pH 7.4 to a concentration of Aβ(1-42) 0.2 mg/ml of Sample were incubated for 1 hour at ambient temperature. Then 100 μl were analyzed exclusion chromatography (SEC).

Aβ(1-42), 70% HCOOH:

1 mg of Aβ(1-42) was dissolved in 50 ál of 70% HCOOH in H2O and stirred for 1 min. at a temperature surrounding the it environment. The sample was centrifuged for 5 min at 10000 g. Collected supernatant. The concentration of Aβ(1-42) in the supernatant was determined according to the method of Bradford (BIO-RAD).

The sample for 5 min:

2 μl of Aβ(1-42) in 70% HCOOH was diluted to a concentration of 0.2 mg/ml Aβ(1-42) using 20 mm NaH2PO4, 140 mm NaCl, pH 7.4 and brought the pH to 7.4 using 1 M NaOH. The sample was incubated for 5 min at ambient temperature. Then 100 μl were analyzed by exclusion chromatography.

The sample for 1 hour:

2 μl of Aβ(1-42) in 70% HCOOH was diluted to a concentration of 0.2 mg/ml Aβ(1-42) using 20 mm NaH2PO4, 140 mm NaCl, pH 7.4 and brought the pH to 7.4 using 1 M NaOH. The sample was incubated for 1 hour at ambient temperature. Then 100 μl were analyzed by exclusion chromatography.

Aβ(1-42), 0.1% of NaOH:

1 mg of Aβ(1-42) (Bachem, catalog # H-1368) were dissolved in 500 μl of 0.1% NaOH in H2O and stirred for 1 min. at ambient temperature. The sample was centrifuged for 5 min at 10000g. Collected supernatant. The concentration of Aβ(1-42) in the supernatant was determined according to the method of Bradford (BIO-RAD).

The sample for 5 min:

20 μl of Aβ(1-42) in 0.1 % NaOH diluted to a concentration of Aβ(1-42) 0.2 mg/ml with 20 mm NaH2PO4, 140 mm NaCl, pH 7,4. The sample was incubated for 5 min at ambient temperature. Then 100 μl were analyzed by exclusion chromatography.

The sample for 1 hour:

20 μl of A is(1-42) in 0.1% NaOH diluted to a concentration of 0.2 mg/ml Aβ(1-42) using 20 mm NaH 2PO4, 140 mm NaCl, pH 7,4. The sample was incubated for 1 hour at ambient temperature. Then 100 μl were analyzed by exclusion chromatography.

Aβ(1-40), with 0.1% NaOH:

1 mg of Aβ(1-40) (Bachem, catalog # H-1194) was dissolved in 500 μl of 0.1% NaOH in H2O and stirred for 1 min. at ambient temperature. The sample was then centrifuged for 5 min at 10000g. Collected supernatant. The concentration of Aβ(1-42) in the supernatant was determined according to the method of Bradford (BIO-RAD).

The sample for 5 min:

20 μl of Aβ(1-40) in 0.1 % NaOH diluted to a concentration of 0.2 mg/ml Aβ(1-40) using 20 mm NaH2PO4, 140 mm NaCl, pH 7,4. The sample was incubated for 5 min at ambient temperature. Then 100 μl were analyzed by exclusion chromatography.

The sample for 1 hour:

20 μl of Aβ(1-40) in 0.1% NaOH diluted to a concentration of 0.2 mg/ml Aβ(1-40) using 20 mm NaH2PO4, 140 mm NaCl, pH 7,4. The sample was incubated for 1 hour at ambient temperature. Then 100 μl were analyzed by exclusion chromatography.

Conditions exclusion chromatography (SEC):

Column SEC:

Superose 12 HR 10/300 GL (Amersham, catalog No. 17-5173-01)

Duct: 0.5 ml/min

Loading paper: 0.2 cm/min

The absorption at 214 nm: 0 to 0.2 units absorption

Mobile phase: 20 mm NaH2PO4, 140 mm NaCl, pH 7,4

The results are shown in figure 1.

Obtaining a solution of the pure monomial is REGO Aβ is a difficult task because of the strong tendency of Aβ peptide, especially monomer Aβ(1-42), to aggregate into fibrils. However, for screening and characterization of antiglobulin Aβ(20-42), a Discerner of the monomers of Aβ(1-42) and Aβ monomers(1-40), you should use the best of technically achievable drugs Monomeric Aβ. Here tested the effect of the initial solvent of the Aβ peptide, the effect of aggregation after further dilution in 20 mm NaH2PO4, 140 mm NaCl, pH 7,4. Supplier of Aβ peptide (Bachem) points out in his technical information that Aβ(1-42) are dissolved in 0.1% NH4OH. After five minutes at room temperature (RT) after dissolution of Aβ(1-42) in NH4OH and immediate further diluted 1:10 in 20 mm NaH2PO4, 140 mm NaCl, pH 7,4 exclusion chromatography showed the first signs of aggregation of Aβ(1-42) to the precursors of fibrils with a minor peak CD. Monomeric Aβ(1-42) is held as the main peak of 11 kDa and shoulder kDa. After incubation for one hour at room temperature (RT) peptide Aβ(1-42) in NH4OH already aggregates to the high content of fibrils of Aβ(1-42), which leads to the loss of the subject detection material, which is not included in the column for exclusive chromatography. If 70% formic acid is used as the initial solvent for the peptide Aβ(1-42)aggregation to a high degree after 1 hour at RT is only for a minor fraction of the OST is segosa monomer Aβ(1-42) (it should be noted, that formic acid by itself leads to high background absorption at the wavelength for detection of protein). The best initial solvent for Aβ(1-42) for the prevention of aggregation is 0.1% NaOH, for which even after 1 hour incubation at dissolution and further breeding showed only a minor fraction of aggregated Aβ(1-42) in the majority of Aβ(1-42), still remaining monomer. For Aβ(1-40), initially dissolved in 0.1% NaOH, never showed signs of aggregation even after 1 hour incubation at RT.

Example 3: Semiquantitative analysis of discrimination fibrils Aβ(1-42) antibody selective for globular Aβ(20-42), with visualization in SDS-PAGE

Getting fibrils Aβ(1-42):

1 mg of Aβ(1-42) (Bachem, cat. no.: H-1368) were dissolved in 500 μl of 0.1% NH4OH in H2O and stirred for 1 min at ambient temperature. The sample was centrifuged for 5 min at 10000 g. Collected supernatant. The concentration of Aβ(1-42) in the supernatant was determined according to the method of Bradford (BIO-RAD).

100 μl of Aβ(1-42) in 0.1% NH4OH was mixed with 300 μl of 20 mm NaH2PO4, 140 mm NaCl, pH 7.4 and brought the pH to 7.4 with 2% HCl. The sample was then incubated at 37°C for 20 hours. Then the sample was centrifuged for 10 min at 10000 g. The supernatant was decanted, and the residue was mixed with 400 μl of 20 mm NaH2PO4, 140 mm NaCl, pH 7,4, resuspending and vigorous stirring ("shaking") for 1 min and centrifuged for 10 min at 10000g. The supernatant was decanted, and the residue was mixed with 400 μl of 20 mm NaH2PO4, 140 mm NaCl, pH 7,4, resuspendable vigorous stirring ("shaking") for 1 min and centrifuged for 10 min at 10000g again. The supernatant was decanted. Sediment resuspendable in 380 μl of 20 mm NaH2PO4, 140 mm NaCl, pH 7.4, which was vigorous stirring ("shaking").

The binding of antibodies against Aβ with fibrils of Aβ(1-42):

40 μl of the preparation of fibrils of Aβ(1-42) was dissolved 160 μl of 20 mm NaH2PO4, 140 mm NaCl, 0,05% Tween 20, pH 7.4 and stirred 5 min at ambient temperature, then the sample was centrifuged 10 min at 10000g. The supernatant was decanted, and the precipitate resuspendable in 95 μl of 20 mm NaH2PO4, 140 mm NaCl, 0,05% Tween 20, pH of 7.4. Re-suspension which was vigorous stirring ("shaking").

Each aliquot of 10 µl of the preparation of fibrils was mixed with:

a) 10 μl of 20 mm NaH2PO4, 140 mm NaCl, pH 7,4;

b) 10 μl of 0.5 μg/μl 5F7 20 mm NaH2PO4, 140 mm NaCl, pH 7,4;

c) 10 μl of 0.5 μg/μl 6E10 (Signet Nr.: 9320) in 20 mm NaH2PO4, 140 mm NaCl, pH 7,4.

Samples were incubated at 37°C for 20 hours, then centrifuged 10 min at 10000g. Supernatant collected and mixed with 20 µl of sample buffer for SDS-PAGE. The remains were mixed with 50 μl of 20 mm NaH2PO4, 140 mm NaCl, 0.025% of Tween 20, pH 7.4 and resuspendable "shaking", and then the samples centrif who was garofali 10 min at 10000 g. Supernatant decanted and the sediments were mixed with 20 μl of 20 mm NaH2PO4, 140 mm NaCl, 0.025% of Tween 20, pH of 7.4, and then 20 μl of sample buffer for SDS-PAGE. Samples were applied to 4-20% Tris/glycine gel electrophoresis.

The parameters for SDS-PAGE:

The sample buffer with SDS: 0.3 g SDS

4 ml 1 M Tris/HCl pH 6,8

8 ml of glycerol

1 ml 1% bromophenol blue in ethanol

Supplement H2O to 50 ml

4-20% Tris/glycine gel: (Invitrogen, cat. no.: EC6025BOX)

Buffer for electrophoresis: 7.5 g Tris

36 g of glycine

2.5 g SDS

Supplement H2O to 2.5 liters

The electrophoresis gel was performed at a constant current of 20 mA.

Staining of gels: Kumasi blue R250

The results are shown in figure 2.

Semiquantitative analysis of various Aβ antibodies and their separation of fibrils of Aβ(1-42). The provisions of antibodies, fibrils Aβ(1-42) and Aβ monomers(1-42) is marked along the edge of the gel. Because of their size fibrils of Aβ(1-42) can not enter the gel for SDS-PAGE, and they can be seen in the wells of the gel.

1. Token.

2. The preparation of fibrils of Aβ(1-42); the control.

3. The preparation of fibrils of Aβ(1-42); + mAb 5F7; 20 h at 37°C; the supernatant.

4. The preparation of fibrils of Aβ(1-42); + mAb 5F7; 20 h at 37°C; precipitation is present.

5. The preparation of fibrils of Aβ(1-42); + mAb 6E10; 20 h at 37°C; the supernatant.

6. The preparation of fibrils of Aβ(1-42); + mAb 6E10; 20 h at 37°C; precipitation is present.

Relative binding to the type of Aβ fibrils was assessed by the analysis in SDS-PAGE by measuring the values of optical density improvement and (OD) for the heavy chains of the antibodies associated with fibrils (sediment fraction) and supernatant fractions after centrifugation. Antibodies that bind to Aβ fibrils, must be deposited with Aβ fibrils and, thus, they are found in the fractions of sediment, while not associated with Aβ fibrils (free) antibodies to be detected in the supernatant. The percentage of antibodies bound to Aβ fibrils, are calculated according to the following formula:

The percentage of antibodies bound to the fibrils Aβ =

ODfraction of fibrils× 100%/(ODfraction of fibrils+ ODthe supernatant fraction).

This method worked for mAb 6E10 (Signet, div. no.: 9320), 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1.

In the brain in Alzheimer's disease Aβ fibrils represent a major component of the total pool of Aβ peptides. Due to the attack of these fibrils antibodies against Aβ increases the risk of negative side effects due to the release of large quantities of Aβ, which can then increase the risk of microchromosome. The increased risk of microchromosome watched for a method of active immunization with fibrillar aggregates of Aβ peptide (Bennett and Holtzman, 2005, Neurology, 64, 10-12; Orgogozo J, Neurology, 2003, 61,46-54; Schenk et al., 2004, Curr Opin Immunol, 16, 599-606).

In contrast to the commercially available antibody 6E10 (Signet 9320)that recognize linear Aβ-epitope between AA1-17, voting for globular Aβ(20-42) antibody 5F7 (which in fact has the lowest selectivity for globalmenu Aβ(20-42) compared with other forms of Aβ) n is associated with the fibrils of Aβ(1-42) in the experiment of co-precipitation. This is demonstrated by the fact that the antibody 5F7 after incubation with fibrils of Aβ(1-42) after the stage of deposition remains in the supernatant and not seacadets due to binding to fibrils of Aβ(1-42).

Example 4: Analysis of cognitive behavior in mice through a test of object recognition after active immunization monomer Aβ(1-42) (0,1% NH4OH, globularia Aβ(1-42) or globularia Aβ(20-42) compared to wild type.

In these experiments used mice, overexpressing APP man with a point mutation. Point mutation refers to the amino acid 717 (substitution of valine for isoleucine) and found in the family from London, where she was led to the debut of the AD before the sixth decade of life (Mullan et al., Nature Genetics 2 (1992) 340-342). Transgenic mice, designated here as APP/L as received and was first described in Leuven (Moechars et al., J. Biol. Chem. 274 (1999) 6483-6492). Female mice APP/L were subjected to active immunization at the age of 6 weeks.

Mice were administered 100 μg of monomer Aβ(1-42) (0.1 % of NH4OH), or globular Aβ(1-42), or globular Aβ(20-42) in phosphate-buffered saline (PBS), mixed with an equal amount of complete adjuvant's adjuvant intraperitoneally, with subsequent booster injections of the same amount of antigen in incomplete Freund's adjuvant every three weeks for three months. During the time period of the experiment, mice were kept in standard conditions of the draws of the day/night cycle (14 hours of light, since 19-00/10 hours dark). The increase in body mass during the time of the experiment was as expected and did not differ from the control group, which was introduced only PBS/adjuvant, suggesting good tolerability of treatments antigen.

At the age of 4.5 months cognitive ability of mice were tested through the test object recognition, as described in this area (Dewachter et al. Journal of Neuroscience 22 (2002) 3445-3453). With this purpose, mice were habituated to the site and then within 10 minutes was subjected to the learning phase, during which the individual was placed on the site now contains two identical items (blue pyramid, green cubes, yellow cylinders of similar size, approximately 4 cm). Recorded the duration and the frequency at which the mouse was investigated objects. During phase memory, 2.5 hours later, the mice were returned to the site now contains, in addition to well-known object unknown object, randomly selected from other objects. The recognition of a new object was recorded as the time during which the mouse was investigated new object in relation to the total time (the study of the old and the new object). "Identification rate" expresses this relationship (time for a new object/total time). A mouse that does not remember the object is known, will assume e is about the same interest, as a new object, and spend the same amount of time on his research, in other words, to have an identification rate of 50%. The mouse that stores the object is known, it will be treated as uninteresting and, therefore, have a significantly higher rate of detection. It is known that mouse APP/L have cognitive impairment at the age of 4.5 months and have identification rate when measured at the level of random, ie 50%.

The results are shown in figure 3.

The test object recognition for mice. The test describes the known object recognition compared with an unknown object, measured in translation on exploratory behavior within 10 minutes of the test phase. The identification rate is defined as the percentage of time that the mouse spent on the study of the unknown object in relation to the time spent on exploring both objects. Mouse explored the known object within a 10-minute phase of training for three hours before the test phase. Compared five groups of mice (number n is given under the columns). Normal mice C57BI/6 (wild type) had high R1 that is significantly different from random (50%, i.e. equal to the time of the study both known and unknown object) (*** = p < 0,001; t-test t-test). Four other groups of transgenic APP mice were subjected to and the active immunization three months earlier. Used immunogen was a monomer Aβ(1-42), globular Aβ(1-42) and globular Aβ(20-42). Phosphate-saline buffer (PBS) was used as control. Significant differences between PBS and other groups are indicated by circles: ° = p < 0,05; °° = p < 0,01 (secondary t-test after p < 0,05 in ANOVA).

It is known that mouse APP/L have, unlike nereshennyh mice, cognitive impairment at the age of 4.5 months, with results close to chance level (i.e. 50% identification rate). In fact, PBS treated mice have a random behavior, in contrast to nereshennyh mice (wild type). Immunization with native globularia Aβ(1-42), as well as globularia Aβ(20-42), leads to significantly improved object recognition in mice APP/L.

Both drug globularia (native and truncated) result in improved memory in transgenic animals APP and even superior recognition in animals treated with globularia Aβ(20-42), it is reasonable to conclude that the induction of antibodies against a truncated globular Aβ(20-42) will lead to better results and that passive immunization with antibodies specifically reactive with these views, a represents the optimal treatment strategy.

Example 5: Dot-blot analysis of the antibody profile for various forms of Aβ after active immunization of mice APP/L Tg-globularia β(20-42).

After immunization of mice (compare example 4) APP/L (Moechars et al., 1999, J. Biol. Chem. 274, 6483-6492) different forms of Aβ in the plasma samples was assessed by antibodies against Aβ. To this end, he got a serial dilution of the forms of Aβ(1-42) in the range of 100 pmol/μl to 0.01 pmol/μl in PBS, supplemented with 0.2 mg/ml BSA. 1 ál of each sample was put points on the nitrocellulose membrane. For detection used the corresponding plasma samples mouse (diluted 1:400). Immunoablative were performed using alkaline phosphatase conjugated antibody against mouse IgG and coloring reagent NBT/BCIP.

Standards Aβ for dot-blotting:

1. globular Aβ(1-42)

Getting globular Aβ(1-42) described in example 1a.

2. HFIP pretreated monomer Aβ(1-42) in Pluronic F68

3 mg of Aβ(1-42), (Bachem Inc.; cat. no. H-1368) were dissolved in 0.5 ml of HFIP (suspension 6 mg/ml) in 1.7 ml Eppendorff tube and shaked (Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37°C to obtain a clear solution. The sample was dried in the SpeedVac concentrator (1.5 hours) and resuspendable 13.2 μl DMSO, shaken for 10 sec, followed by sonication (20 s), and shaked (for example, in Eppendorff Thermo mixer, 1400 rpm) for 10 minutes was Added 6 ml of 20 mm NaH2PO4; 140 mm NaCl; 0.1% Pluronic F68; pH 7.4 and was stirred for 1 hour at room temperature. The sample was centrifuged who for 20 min at 3000 g. The supernatant was removed and the precipitate was dissolved in 0.6 ml of 20 mm NaH2PO4; 140 mm NaCl; 1 % Pluronic F68, pH of 7.4. Added 3.4 ml of H2O and stirred for 1 hour at room temperature followed by centrifugation for 20 min at 3000g. Eight aliquot from each 0.5 ml of the supernatant was kept at -20°C for further use.

3. Globular Aβ(20-42)

Getting globular Aβ(20-42) described in example 1c.

4. Globular Aβ(12-42)

Getting globular Aβ(12-42) described in example 1d.

5. HFIP pretreated monomer Aβ(1-40), 5 mm in DMSO

1 mg of Aβ(1-40), (Bachem Inc., cat. # H-1194) suspended in 0.25 ml of HFIP (suspension 4 mg/ml) in an Eppendorff tube. The tube was shaken (for example, in Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37°C to obtain a clear solution and then dried in the hub speed vac (1.5 h). The sample was re-dissolved in 46 ál DMSO (21,7 mg/ml = 5 mm)was shaken for 10 sec and then treated with ultrasound for 20 sec. After 10 min shaking (for example, in Eppendorff Thermo mixer, 1400 rpm), the Sample was kept at -20°C for further use.

6. The monomer Aβ(1-42), 0.1% of NH4OH

1 mg of Aβ(1-42) (Bachem Inc., cat. # H-1368) were dissolved in 0.5 ml of 0.1% NH4OH in H2O (freshly prepared) (= 2 mg/ml) and immediately shaken for 30 sec at room temperature to obtain a transparent solution. The sample is kept our at -20°C for further use.

7. The fibrils of Aβ(1-42)

1 mg of Aβ(1-42) (Bachem Inc. catalog # H-1368) were dissolved in 500 ál of water with 0.1% NH4OH (Eppendorff tube), and the sample was stirred for 1 min at room temperature. 100 μl of this freshly prepared solution of Aβ(1-42) was neutralized with 300 μl of 20 mm NaH2PO4; 140 mm NaCl, pH 7,4. the pH was brought to a pH of 7.4 with 1% HCl. The sample was incubated for 24 h at 37°C and centrifuged (10 min at 10000g). The supernatant was removed and the precipitate fibrils resuspendable in 400 μl of 20 mm NaH2PO4; 140 mm NaCl, pH 7,4 by shaking for 1 minute

8. sAPPα

Supplied from Sigma (cat. No. S9564; 25 μg in 20 mm NaH2PO4; 140 mm NaCl; pH 7,4). sAPPα were diluted in 20 mm NaH2PO4, 140 mm NaCl, pH of 7.4, 0.2 mg/ml BSA 0.1 mg/ml (= 1 pmol/ál).

Materials for the dot-blot

Standards Aβ:

Serial dilution of Aβ antigens in 20 mm NaH2PO4, 140 mm NaCl, pH 7,4+ 0,2 mg/ml BSA

1) at 100 pmol/μl;

2) 10 pmol/μl;

3) 1 pmol/μl;

4) 0.1 pmol/μl;

5) 0.01 pmol/μl.

Nitrocellulose:

The environment for migration TRANS-blot, pure nitrocellulose membrane (0.45 µm); BIO-RAD

Anti-mouse-AP:

AQ330A (Chemicon)

Reagent for detection:

Tablets NBT/BCIP (Roche)

Bovine serum albumin (BSA):

A-7888 (SIGMA)

Blocking reagent:

5% low-fat milk in TBS

Buffer solutions:

TBS

25 mm Tris/HCl buffer pH 7.5

+ 150 mm NaCl

TTB

25 mm Tris/HCl buffer pH 7.5

+ 150 mm NaCl

+ 0,05 % Tween 20

PBS + 0.2 mg/ml BSA

20 mm buffer NaH2PO4a pH of 7.4

+ 140 mm NaCl

+ 0.2 mg/ml BSA

A solution of antibodies I:

Plasma samples mouse from studies of active immunization globularia Aβ(20-42) (dilution 1:400 in 20 ml of 1% milk with a low fat content in TBS)

The antibody solution II:

a dilution of 1:5000

Anti-mouse-AP in 1% milk with a low fat content in TBS

The way the dot-blotting:

1) 1 ál each of the different standards Aβ (5 serial dilutions) were marked points on the nitrocellulose membrane at a distance of approximately 1 cm from each other.

2) Points standards Aβ allowed to dry on the nitrocellulose on the air for at least 10 min at room temperature (RT) (= dot-blot)

3) Blocking:

The dot blot was incubated with 30 ml of 5% milk with a low fat content in TBS for 1.5 h at RT.

4) Washing:

The blocking solution was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT.

5) Solution of antibodies I:

The buffer wash was decanted, and the dot blot incubated with antibody solution I over night at RT.

6) Washing:

The solution I antibodies was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The rinse solution was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min Ave is RT. The rinse solution was decanted and the dot blot incubated under shaking with 20 ml TBS for 10 min at RT.

7) the antibody Solution II:

The buffer wash was decanted, and the dot blot incubated with antibody solution II for 1 h at RT

8) Washing:

The antibody solution II was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The rinse solution was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The rinse solution was decanted, and the dot blot incubated under shaking with 20 ml TBS for 10 min at RT.

9) the method:

The rinse solution was decanted. 1 tablet NBT/BCIP was dissolved in 20 ml of H2O, and the dot blot incubated for 5 min with this solution. The development was stopped by intense washing H2O.

The results are shown in figure 4.

Analysis of dot-blotting antibodies against Aβ received after active immunization of mice with globularia Aβ(20-42) to assess their specificity towards different forms of Aβ. Selected forms of Aβ put points in serial dilutions and incubated with the appropriate mouse plasma containing antibodies against Aβ obtained during the immune response. Separate dot-blots correspond to different individuals immunized mice.

1. Globular Aβ(1-42).

2. The monomer Aβ(1-42), HFIP pretreated in 0.1% Pluronic F68.

3. Globular Aβ(20-42).

4. Globular Aβ(12-42).

5. The monomer Aβ(1-40), HFIP pretreated with 5 mm in DMSO.

6. The monomer Aβ(1-42), 0.1% of NH4OH.

7. The preparation of fibrils of Aβ(1-42).

8. sAPPα (Sigma); (the first point: 1 pmol).

In the study of active immunization of mice APP/L Tg showed that immunization with globularia Aβ(20-42) leads to better results to facilitate cognitive impairment in these mice compared with PBS treatment. For plasma samples from mice APP/L Tg after active immunization of globalmenu Aβ(20-42) showed the antibody profile (preferred recognition globular Aβ(20-42) and globular Aβ(12-42), resembling the profile of mAb against globular Aβ(20-42)stated herein.

Example 6: the Concentration of soluble and insoluble Aβ peptide(1-42) and Aβ(1-40) in brain extracts of mice actively immunized APP/PS1 Tg monomer Aβ(1-42), globularia Aβ(1-42), globularia Aβ(20-42) or medium as a control.

40 female mice from double-transgenic model of Alzheimer's disease in mice (mouse APP/PS1 Tg) on the background FVBxC57BI/6J at the age of 4 months were used for this study. Mouse APP/PS1 Tg have a 695 amino acid form of an APP person with V717I mutation (position in relation to the most long isoforms of APP) and, in addition, gene penicilina 1 person with a mutation A264E. Both genes are under the control of the Thy1 promoter. Mice received characte savali in one of the main laboratories reMYND, Experimental Genetics Group, Campus Gasthuisberg, Catholic University Leuven, by Prof. Fred Van Leuven et al.

All mice were genotypically by polymerase chain reaction (PCR) at the age of 3 weeks and were assigned a unique identification number, as soon as it became known PCR results.

Mice had free access to a pre-filtered and sterile water (UV lamp) and standard diet to mice. Food kept in dry and cold conditions in a well-ventilated store. The amount of water and feed was checked daily, was added as necessary by default updated twice a week.

Mice contained when facing the rhythm of day and night: 14 hours light/10 hours dark since 19-00, in standard metal cages type RVS T2 (area 540 cm2). Cells are equipped with solid floors and a layer of straw bedding. The number of mice per cage were restricted in accordance with the legislation on animal welfare. Five days before the start of the test the behavior of the mice were placed in other cells Makrolon type 2 and transported to the laboratory for adaptation to the laboratory environment when preparing for the test behavior.

Mice were administered 100 μg of monomer Aβ(1-42) (0.1% of NH4OH, globular Aβ(1-42) or globular Aβ(20-42) in phosphate-buffered saline (PBS), mixed with an equal amount of complete adjuvant's adjuvant intraperitoneally, with the second booster injection of the same amount of antigen in incomplete Freund's adjuvant every three weeks for four months.

Biochemistry

Aβ(1-40) and Aβ(1-42) in the soluble fraction of 1 hemisphere of the brain was determined by ELISA. In addition, Aβ(1-40) and Aβ(1-42) in the insoluble membrane fraction of 1 hemisphere of the brain was determined by ELISA.

Mice were anestesiologi a mixture of 2:1:1 Ketalar (ketamine), Rompun (xylazine 2%) and atropine and held transcardially washing physiological serum at 4°C. This was performed to remove blood from the vessels of the brain, procedures that do not affect the integrity of the bodies. Cerebrospinal fluid (CSF) was collected by incision of the muscles between the skull and the first cervical vertebra. Did puncture in mostomozzhechkovogo tank needle size 26 and 10-20 µl, CSF was collected with a fine glass pipette.

Blood was collected by heart puncture, and 1 ml was collected by syringe coated with heparin tubes Eppendorf. The blood was centrifuged at 14000 rpm at 4°C for 5 minutes. Serum was stored at -70°C.

Mice were performed transcardially washing physiological serum at 4°C.

The brain was removed from the skull, the back of the brain and forebrain were separated by incision in the coronary/frontal plane. The cerebellum was removed. The forebrain is divided into left and right hemisphere using an incision in the midline.

One hemisphere was immediately immersed in liquid nitrogen and kept at -70°C until biochemical analysis is.

Homogenization and fractionation of the hemisphere of the brain

The brain is homogenized using a Potter homogenizer, glass vials (free of detergent, 2 cm3and mechanical homogenizer (650 rpm). The volume of 6.5 × ½ of the brain mass of freshly prepared 20 mm buffer Tris/HCl (pH 8.5) with protease inhibitors (1 tablet per 50 ml of buffer Tris/HCl, Complete™, Roche, Mannheim, Germany) was used as buffer for homogenization.

Samples transferred to -70°C in the holder for samples with liquid nitrogen, and each sample was pre-heated by incubating on the table for a few seconds before homogenization. The homogenates were collected in centrifuge tubes Beckman TLX and kept in ice before centrifugation. Between the two samples homogenizer (Potter and the glass test tube was thoroughly washed with distilled water (AD) without detergent and dried with absorbent paper.

Samples were centrifuged in a pre-chilled the ultracentrifuge (Beckman, Mannheim, Germany) for 1 hour and 20 minutes at 48000 rpm (the 135,000 × g) at 4°C. because of the limited number of holders in the centrifuge (N=8) samples were sorted by weight of the brain (for balancing centrifuge) and distributed randomly to separate different groups of processing on different sessions centrifugation.

The supernatant (Astoria fraction, containing secreted APP and amyloid peptides were separated from the sediment (membrane-containing fractions associated with membrane fragments of APP and associated with plaque amyloid peptides in the case of old mice). The supernatant was divided into two tubes, of which one was kept at -20°C in reserve, and the other further processed by chromatography on a column for the concentration of amyloid peptides.

The mass of the brain, the volume of used buffer Tris/HCl, sessions centrifugation (color coded) and the volume of the soluble fraction was used for chromatography on the column, shown as an example in the following table.

No. sampleProcessingN° ID mouseThe mass of the brain (W) (mg)V Tris (=W×6,5)
(ál)
50% V Tris.
(ál)
19XTAB.TPF 1305157,81026513
21XTAB.TPF 1335160,21041521

Small speakers with education the military phase (C18-Sep-Pack Vac 3cc cartridges, Waters, Massachusetts, MA) was installed in the vacuum system and washed with 80% acetonitrile in 0.1% triperoxonane acid (TFA) followed by two washes with 0.1% TFA. Then the samples were applied to the column, and the column is sequentially washed with 5% and 25% A-TFA. The peptides of amyloid was suirable 75% A-TFA, and eluate was collected in 2 ml tubes in ice. Eluate liofilizirovanny in a SpeedVac concentrator (Savant, Farmingdale, NY) during the night and re-dissolved in 330 µl of diluent supplied with the kits ELISA.

Precipitation further fractionally on various membrane fractions: membrane fraction A (MFA), membrane fraction B (MFB), containing the full APP and membrane fraction C (MFC)containing associated with amyloid plaques. Thus, the samples were dissolved in TBS buffer with protease inhibitors (1 tablet per 50 ml buffer TBS, Complete™, Roche, Mannheim, Germany), and MFA was divided into two tubes, of which one was kept at -20°C in reserve. 60% MFA was further processed by addition of NP40 (2% of final volume) and Triton X-100 (2% of final volume) in TBS with protease inhibitors and centrifuged for one hour at 27000 rpm (98000 × g) in a Beckman ultracentrifuge at 4°C using a swing rotor (SW60). The supernatant (MFB) was separated from the sediment (MFC), and both were stored at -20°C.

The mass of the brain, 60% by weight of the brain, the volume of used buffer TBS + PI + NP40 + Triton X-100 and sessions center is pulirovaniya (color coded) are shown as an example in the following table.

No. sampleProcessingN° ID mouseThe mass of the brain (W) (mg)3/5 × the mass of the brain
(mg)
Buffer size = 3/5 W × 15 (mm)
19XTAB.TPF 1305157,8951420
21XTAB.TPF 1335160,2961442

Aβ ELISA human soluble fraction of one hemisphere

To evaluate the amount of Aβ(1-40) human and Aβ(1-42) human rights in the soluble fraction of brain homogenates and/or in cerebrospinal fluid (CSF) used commercially available kits for enzyme-linked immunosorbent assay (ELISA) (highly sensitive ELISA for amyloid h β40 or β42, The Genetics Company, Zurich, Switzerland). ELISA was performed according to the Protocol of the manufacturer. Briefly, standards (cultivation of synthetic Aβ(1-40) or Aβ(1-42)) and the samples were prepared in 96-well polypropylene tablet without the ability of the binding protein (Greiner bio-one, Frickenhausen, Germany). Standard cultivation with konecne the mi concentrations of 1000, 500, 250, 125, 62,5, 31,3 and 15.6 PG/ml and the samples were received in the diluent supplied with the ELISA kit, in the final volume of 60 ál. Since the levels of amyloid increases with the age of the mouse and as for the true evaluation is necessary to read the samples were within the linear portion of the standard curve, samples for analyses of Aβ(1-40) was diluted 1:3, samples for analyses of Aβ(1-42) was diluted 1:6.

Samples, standards and blank controls (50 ál) was applied to coated with anti-Aβ polystyrene tablet (with a fixed antibody selectively recognizes the C-end-of-antigen) in addition to the election conjugate antibodies against Aβ (biotinylated antibody for detection), and incubated overnight at 4°C to allow formation of a complex of the antibody-amyloid-antibody. The next day was added to the conjugate streptavidin-peroxidase followed by the addition after 30 minutes the mixture TMB/peroxide, which leads to the transformation of the substrate into a colored product. This reaction was stopped by adding sulfuric acid (1 M), and the colour intensity was measured by photometry using the reader for ELISA with 450 nm filter. Quantification of Aβ content in the samples was obtained by comparing the absorbance with the standard curve obtained with synthetic Aβ(1-40) or Aβ(1-42).

Aβ ELISA person in the insoluble fraction of the Noah hemisphere

To evaluate the amount of Aβ(1-40) human and Aβ(1-42) man in the insoluble membrane fraction of homogenates of brain MFC samples were additionally processed and dissolved in the 8M guanidine in 80 mm Tris/HCl. Then the samples were incubated for 3 hours in termoustoychivee at 25°C and pietravalle up and down using a 100 μl pipette every hour to dissolve the precipitate MFC in guanidine buffer. Finally, the samples were centrifuged for 1 minute at 4000 rpm to remove debris.

The mass of the brain, mass of sediment MFC and the volume of 8 M guanidine buffer is shown as an example in the following table.

No. sampleProcessingN° ID mouseThe mass of the brain (W) (mg)The mass of sediment MFC (WMFC) (40%)Volume of 8 M guanidine (WMFC x 1.6) (μl)
19XTAB.TPF 1305157,863101
21XTAB.TPF 1335160,264103

To evaluate the amount of Aβ(1-40)human and Aβ(1-42) human rights in finite samples, used commercially available kits for enzyme-linked immunosorbent assay (ELISA) (highly sensitive ELISA for amyloid h β40 or β42 ELISA, The Genetics Company, Zurich, Switzerland). ELISA were performed according to the manufacturer's Protocol, except with the prior standards (cultivation of synthetic Aβ(1-40) or Aβ(1-42)). The samples were received in the diluent supplied with the ELISA kit, in the final volume of 60 ál. Since guanidine affect the values of the standard curve OD, received standard cultivation with final concentrations 1000, 500, 250, 125, 62,5, 31,3 and 15.6 PG/ml was obtained in the diluent for samples with the same concentration of guanidine, as in the samples. It was performed in 96-well polypropylene tablet without the ability of the binding protein (Greiner bio-one, Frickenhausen, Germany).

Since the levels of amyloid increases with the age of the mouse and as for the true evaluation is necessary to read the samples were within the linear portion of the standard curve, samples for analyses of Aβ(1-40) was diluted 1:3, samples for analysis of insoluble Aβ(1-40) and insoluble Aβ(1-42) was diluted 1:500.

Samples, standards and blank controls (50 ál) was applied to coated with anti-Aβ polystyrene tablet (with a fixed antibody selectively recognizes the C-end-of-antigen) in addition to the election conjugate antibodies against Aβ (biotinylated ant the body for detection), and incubated overnight at 4°C, to allow formation of a complex of the antibody-amyloid-antibody. The next day was added to the conjugate streptavidin-peroxidase followed by the addition after 30 minutes the mixture TMB/peroxide, which leads to the transformation of the substrate into a colored product. This reaction was stopped by adding sulfuric acid (1 M), and the colour intensity was measured by photometry using the reader for ELISA with 450 nm filter. Quantification of Aβ content in the samples was obtained by comparing the absorbance with the standard curve obtained with synthetic Aβ(1-40) or Aβ(1-42).

The results are shown in figure 5

The concentration of soluble and insoluble Aβ peptide(1-42) and Aβ peptide(1-40) in extracts of brain actively immunized mice, APP/PS1 Tg monomer Aβ(1-42) (0.1% of NH4OH, globularia Aβ(1-42), globularia Aβ(20-42) or medium as a control.

In soluble and insoluble fractions of the extract of mouse brain APP/PS1 Tg, actively immunized by globularia Aβ(20-42), the level of peptides Aβ(1-40) and Aβ(1-42) were not significantly different from the control carrier. In contrast, immunization with globularia Aβ(1-42) and the monomer Aβ(1-42) leads to a decrease in the brain levels of Aβ(1-40) and Aβ(1-42). This indicates that the method of direct immunization globularia Aβ(20-42) does not significantly alter the overall levels of Aβ in the brain, but, nevertheless, is the effect is effective for the relief associated with Aβ peptide cognitive impairment (see example 4).

Example 7: Analysis of cognitive behavior through the test object recognition for transgenic APP mice/L after passive immunization with antibodies against globular Aβ(20-42).

In these experiments used mice with overexpressing APP man with a point mutation. Point mutation refers to the amino acid 717 (substitution of valine for isoleucine) and found in the family from London, where she was led to the debut of the AD before the sixth decade of life (Mullan et al., Nature Genetics 2 (1992) 340-342). Transgenic mice, designated here as APP/L as received and was first described in Leuven (Moechars et al., J. Biol. Chem. 274 (1999) 6483-6492). Female mice APP/L were subjected to passive immunization at the age of 3 months. The mice were injected with 250 µg of any of the monoclonal mouse antibodies 5F7, 10F11 or 7C6 in 100 μl of phosphate-saline buffer (PBS). During the time period of the experiment animals were kept under standard conditions with reversed day/night cycle (14 hours of light, since 19-00/10 hours dark). They are well tolerated by passive immunization, without any signs of adverse effects.

After the third injection (day 15 of the experiment, the cognitive ability of the mice tested through the test object recognition, as described in this area (Dewachter et al. Journal of Neuroscience 22 (2002) 3445-3453). With this purpose, mice were habituated to the site and then within 10 minutes was subjected to f the se training during which the individual was placed on the site now contains two identical items (green cubes or orange cylinders of similar size, approximately 4 cm). Recorded the duration and the frequency at which the mouse was investigated objects. During phase memory, 2.5 hours later, the mice were returned to the site now contains, in addition to well-known object, another object. The recognition of a new object was recorded as the time during which the mouse was investigated new object in relation to the total time (the study of the old and the new object). "Identification rate" expresses this relationship (time for a new object/total time). A mouse that does not remember the object is known, it will be treated as the same interesting as a new object, and spend the same amount of time on his research, in other words, to have an identification rate of 50%. The mouse that stores the object is known, it will be treated as uninteresting and, therefore, have a significantly higher rate of detection. It is known that mouse APP/L have cognitive impairment at the age of 4.5 months and have identification rate when measured at the level of random, ie 50%.

The results are shown in figure 6.

The test object recognition for mice. The test describes the recognition Izv the local object is compared with the unknown object, measured in translation on exploratory behavior during the 10-minute phase of testing. The identification rate is defined as the percentage of time that the mouse spent on the study of the unknown object in relation to the time spent on exploring both objects. Mouse explored the known object within a 10-minute phase of training for 2.5 hours before the test phase.

a) APP Transgenic mice were immunized once a week for three weeks by intraperitoneal injection of 250 micrograms of antibody 5F7 (n=9), antibody 10F11 (n=11) or antibodies 7C6 (n=11); control animals were injected with PBS (n=6). Significant differences from random level (50%, i.e. equal to the time of the study both known and unknown object) are marked with asterisks. * = p < 0,05 (t-test)

(b) Comparison of all mice treated with antibody 5F7, 10F11 and 7C6; (n=31)and mice treated with phosphate-saline buffer (PBS; n=6). RI-treated antibody group was significantly different from random level (** = P<0,01; t-test).

It is known that mouse APP/L have cognitive impairment at the age of 4.5 months and have identification rate in the scale of values of a random level, ie 50%.

Indeed, for the PBS treated mice showed a random behavior. Passive immunization with all three antibodies (5F7, 10F11 and 7C6) led to markedly increased the rate R is spozywania. When comparing polerowanie group compared with controls, the identification rate is significantly increased. This favorable effect on the performance of memory mice APP/L after administration of all three antibodies suggests that the antibody against a truncated globular Aβ(20-42) is sufficient to achieve cognitive improvement.

Example 8: the Dot-blot analysis of selectivity of antibodies against globular Aβ(20-42).

To characterize the selectivity of monoclonal antibodies against globular Aβ(20-42), they were used as probes to distinguish the various forms of Aβ. To this end, he got a serial dilution of the forms of Aβ(1-42) in the range of 100 pmol/μl to 0.01 pmol/μl in PBS, supplemented with 0.2 mg/ml BSA. 1 ál of each sample was put points on the nitrocellulose membrane. For detection used the appropriate antibody (0.2 ág/ml). Immunoablative were performed using conjugated to peroxidase antibodies against mouse IgG and coloring substrate reagent BM Blue POD (Roche).

Standards Aβ for dot-blotting:

1. The monomer Aβ(1-42), 0.1 % of NH4OH

1 mg of Aβ(1-42) (Bachem Inc., cat. # H-1368) were dissolved in 0.5 ml of 0.1% NH4OH in H2O (freshly prepared) (= 2 mg/ml) and immediately shaken for 30 sec at room temperature to obtain a transparent solution. brezec kept at -20°C for further use.

2. The monomer Aβ(1-40), with 0.1% NH4OH

1 mg of Aβ(1-40) (Bachem Inc., cat. # H-1368) were dissolved in 0.5 ml of 0.1% NH4OH in H2O (freshly prepared) (= 2 mg/ml) and immediately shaken for 30 sec at room temperature to obtain a transparent solution. The sample was stored at -20°C for further use.

3. The monomer Aβ(1-42), a 0.1% NaOH

2.5 mg Aβ(1-42) (Bachem Inc., cat. # H-1368) were dissolved in 0.5 ml of 0.1% NaOH in H2O (freshly prepared) (= 5 mg/ml) and immediately shaken for 30 sec at room temperature to obtain a transparent solution. The sample was stored at -20°C for further use.

4. The monomer Aβ(1-40), with 0.1% NaOH

2.5 mg Aβ(1-40) (Bachem Inc., cat. # H-1368) were dissolved in 0.5 ml of 0.1% NaOH in H2O (freshly prepared) (= 5 mg/ml) and immediately shaken for 30 sec at room temperature to obtain a transparent solution. The sample was stored at -20°C for further use.

5. Globular Aβ(1-42)

Getting globular Aβ(1-42) described in example 1a.

6. Globular Aβ(12-42)

Getting globular Aβ(12-42) described in example 1d.

7. Globular Aβ(20-42)

Getting globular Aβ(20-42) described in example 1c.

8. The fibrils of Aβ(1-42)

1 mg of Aβ(1-42) (Bachem Inc. cat. # H-1368) were dissolved in 500 ál of water with 0.1% NH4OH (Eppendorff tube and the sample was stirred for 1 min at room temperature. 100 μl of this freshly races the thief Aβ(1-42) was neutralized with 300 μl of 20 mm NaH 2PO4; 140 mm aCl, a pH of 7.4. the pH was brought to a pH of 7.4 with 1% HCl. The sample was incubated for 24 h at 37°C and centrifuged (10 min at 10000 g). The supernatant was removed and the residue fibrils resuspendable in 400 μl of 20 mm NaH2PO4; 140 mm NaCl, pH 7,4 by shaking for 1 minute

9. sAPPα

Supplied from Sigma (cat. No. S9564; 25 μg in 20 mm NaH2PO4; 140 mm NaCl; pH 7,4). sAPPα was diluted to 0.1 mg/ml (= 1 pmol/ál) 20 mm NaH2PO4, 140 mm NaCl, pH of 7.4, 0.2 mg/ml BSA.

Materials for dot-blotting:

Standards Aβ:

Serial dilution of Aβ antigens in 20 mm NaH2PO4, 140 mm NaCl, pH 7,4 + 0,2 mg/ml BSA

1) 100 pmol/ál

2) 10 pmol/ál

3) 1 pmol/ál

4) 0.1 pmol/µl

5) 0.01 pmol/ál

Nitrocellulose:

The environment for migration TRANS-blot, pure nitrocellulose membrane (0.45 µm); BIO-RAD

Anti-mouse-POD:

Cat No. 715-035-150 (Jackson lmmuno Research)

Reagent for detection: a Substrate BM Blue POD, precipitating (Roche)

Bovine serum albumin (BSA):

Cat. # A-7888 (SIGMA)

Blocking reagent:

5 % milk with a low fat content in TBS

Buffer solutions:

TBS, 25 mm Tris/HCl buffer pH 7.5

+ 150 mm NaCl

TTBS

25 mm Tris/HCl buffer pH 7.5 + 150 mm NaCl + 0.05 % of Tween 20

PBS + 0.2 mg/ml BSA

20 mm buffer NaH2PO4a pH of 7.4

+ 140 mm NaCl

+ 0.2 mg/ml BSA

A solution of antibodies I:

0.2 ug/ml antibody diluted in 20 ml of 1 % milk with a low fat content in TBS

p> The antibody solution II:

a dilution of 1:5000

Anti-mouse-POD in 1% milk with a low fat content in TBS

The way the dot-blotting:

1) 1 ál each of the different standards Aβ (5 serial dilutions) were marked points on the nitrocellulose membrane at a distance of approximately 1 cm from each other.

2) Points standards Aβ allowed to dry on the nitrocellulose on the air for at least 10 min at room temperature (RT) (=dot-blot).

3) Blocking:

The dot blot was incubated with 30 ml of 5% milk with a low fat content in TBS for 1.5 h at RT.

4) Washing:

The blocking solution was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT.

5) Solution of antibodies I:

The buffer wash was decanted and the dot blot incubated with antibody solution I for 2 h at RT

6) Washing:

The solution I antibodies was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The rinse solution was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The rinse solution was decanted and the dot blot incubated under shaking with 20 ml TBS for 10 min at RT.

7) the antibody Solution II:

The buffer wash was decanted and the dot blot incubated with antibody solution II during the night when RT

8) Washing:

The antibody solution II was decanted and the dot blot incubated the under shaking with 20 ml TTBS for 10 min at RT. The rinse solution was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The rinse solution was decanted and the dot blot incubated under shaking with 20 ml TBS for 10 min at RT.

9) the method:

The rinse solution was decanted. The dot-blot showed using 10 ml of substrate BM Blue POD within 10 minutes of the Development was stopped by intense washing the dot-blot H2O. quantification were performed using densitometric analysis (GS800 densitometer (BioRad) and a software package Quantity one 4.5.0 (BioRad)by intensity pixels. Evaluated only point having a relative density of greater than 20% of the relative density of the latter is uniquely determined point of globular Aβ(20-42). This threshold value is independently determined for each dot-blot independently. The calculated value indicates the ratio between the recognition of globular Aβ(20-42) and the corresponding form of Aβ for this antibody.

The results are shown in figure 7.

Analysis of dot-blotting specificity of different antibodies against Aβ (6E10, 5F7, 4B7, 10F11, 6A2, 4D10, 2F2; 3B10, 7C6, 7E5, 10C1) in relation to various forms of Aβ. Tested monoclonal antibodies obtained (except 6E10) by active immunization of mice with globularia Aβ(20-42) with subsequent selection of merged cells hybridoma. A separate form A is applied in serial dilutions and incubated with the appropriate antibodies for the immune response.

1. The monomer Aβ(1-42), 0.1 % of NH4OH

2. The monomer Aβ(1-40), with 0.1% NH4OH

3. The monomer Aβ(1-42), a 0.1%NaOH

4. The monomer Aβ(1-40), with 0.1% NaOH

5. Globular Aβ(1-42)

6. Globular Aβ(12-42)

7. Globular Aβ(20-42)

8. The preparation of fibrils of Aβ(1-42)

9. sAPPα (Sigma); (the first point: 1 pmol)

Election mAb against globular Aβ(20-42) can be divided into 3 classes against discrimination globular Aβ(1-42) and globular Aβ(12-42). The first class includes antibody 6A2, 5F7 and 2F2, learns preferably globular Aβ(20-42) and, to some extent, globular Aβ(1-42) (and also globular Aβ(12-42)). The second class includes antibody 10F11, 4D10 and 3B10, preferably recognizes globular Aβ(20-42) and also learns globular Aβ(12-42), but to a lesser extent, and do not know considerably globular Aβ(1-42). The third class, which includes antibodies 7C6, 4B7, 7E5 and 10C1 knows globular Aβ(20-42), but it showed no significant recognition. All three classes will not recognize largely Monomeric Aβ(1-42), Monomeric Aβ(1-40)fibrils of Aβ(1-42) or sAPPα.

The selectivity profile of antibodies against globular Aβ(20-42) showed that significantly increased the identification rate in passive immunization (figure 6) should be mainly due to the selective recognition of a truncated globular Aβ(20-42) and globular Aβ(12-42) and to a lesser extent globular Aβ(1-42) and the e Monomeric Aβ(1-42), Monomeric Aβ(1-40)fibrils Aβ(1-42) or sAPPα.

Example 9: Analysis ofin situa specific antibody response, selective for Aβ(20-42), with fibrillar Aβ peptide in the form of Aβ plaques in aged TG2576 mice with amyloid Aβ vessels of the meninges.

For these experiments we used the material brain of TG2576 mice at the age of 19 months (Hsiao et al., 1996, Science; 274(5284), 99-102) or mice APP/LxPS1 at the age of 9 months (description as above; ReMYND, Leuven, Belgium) or autopsy material two patients with Alzheimer's disease (RZ16 and RZ55; obtained from BrainNet, Munich). In mice sverkhekspressiya APP man with the so-called Swedish mutation (K670N/M671L; Tg2576) or the so-called London mutation (V717I), in addition to gene presenilin 1 person with a mutation A264E (APP/LxPS1) and formed deposits of β-amyloid in the brain parenchyma at the age of approximately 7 to 11 months and deposition of β-amyloid in the larger vessels of the brain at the age of approximately 18 months (Tg2576). Animals were subjected to deep anesthesia and transcardially perfusion with 0.1 M phosphate-saline buffer (PBS) for cleaning the blood. Then the brain was removed from the skull and was divided in the longitudinal direction. One hemisphere of the brain was rapidly frozen, others were fixed by immersion in 4% paraformaldehyde. Fixed by immersion hemisphere subjected cryoprotective by wetting in 30% sucrose in PBS and mounted on a frozen microtome Entire forebrain was cut into sections of 40 μm, which were collected in PBS and used for subsequent staining method. The material of the human brain was a deeply frozen block from the neocortex approximately 1 cm3. A small part of the bar were fixed by immersion in 4% paraformaldehyde and then treated as a material of the mouse brain.

Individual slices were stained with Congo red using the following method:

Material:

kit dye for amyloid Congo red (Sigma-Aldrich; HT-60), consisting of an alcoholic solution of NaCl, NaOH and the solution of Congo red;

- cuvette for dyeing;

- slides for microscope SuperfrostPlus and cover glass;

- ethanol, xylene, Wednesday to fill.

Reagents:

- breeding NaOH 1:100 NaCl get alkaline saline;

- breeding alkaline saline solution 1:100 solution of Congo red, get alkaline solution of Congo red (not more than 15 min before use, the filtrate);

- mount sections on a glass slide and allow them to dry;

- incubate the slide in a ditch for painting, during the first 30-40 minutes in an alkaline saline solution, then for 30-40 minutes in an alkaline solution of Congo red;

- washed three times with fresh ethanol and pour xylene.

Staining at first it is and photographed using a microscope Zeiss Axioplan and evaluated quantitatively. Red pointed to the deposition of amyloid in the form of plaques and in the larger vessels of the meninges. These results are shown in Fig. 8A. Further evaluation of the staining antibody has been focusing on these structures.

The antibody staining was performed by incubating the sections with a solution containing 0.07 to 7,0 mg/ml of the respective antibody in accordance with the following method:

Materials:

- rinse solution TBST (buffered by Tricom saline with Tween 20; 10× concentrate; DakoCytomation; S3306 1:10 in pedestrianonly water)

to 0.3% H2O2in methanol;

- donkey serum (Serotec), 5% in TBST;

- monoclonal antibody mouse against globularia diluted in TBST;

secondary antibody: biotinylated antibody donkey antibodies against mouse (Jackson Measurement; 715-065 - 150; diluted 1:500 in TBST);

- StreptABComplex (DakoCytomation; K 0377);

- set with the substrate for the peroxidase diaminobenzidine (=DAB; Vector Laboratories; SK-4100);

objective cover glass for microscope SuperFrost Plus;

- free from xylene environment for fill (Medite; X-tra Kitt).

Method:

- flooding slices ice 0,3% H2O2and incubated for 30 min;

- washed for 5 min in TBST buffer;

- incubated with donkey serum/TBST for 20 minutes.

- incubated with the primary antibody for 24 hours at room temperature;

- washed in TBST buffer for 5 minutes;

- incubated with blocking serum from a set with peroxidase Vectastain Elite ABC for 20 minutes.

- washed in TBST buffer for 5 minutes;

- incubate with secondary antibody for 60 minutes at ambient temperature;

- washed in TBST buffer for 5 minutes;

- incubated with StreptABComplex for 60 minutes at ambient temperature;

- washed in TBST buffer for 5 minutes;

- incubated with DAB set with peroxidase Vectastain Elite ABC for 20 minutes.

- mount sections on slides, dried in air, dehydration their alcohol fix.

In addition to visual inspection staining, staining of plaques, in addition, assessed quantitatively through a graphical cut 10 randomly selected plaques from histological images using image analysis ImagePro 5.0 and determine the average values for grayscale. The values of optical density was calculated from the values in the gray scale by subtracting the average background density of the colored material from the density of amyloid plaques (0% - no staining plaques above the ambient background, 100% no transition/maximum staining), and tested the statistical significance between control and antibodies, as well as between 6G1 and antibodies selective for Aβ(20-42), with the NGOs, using ANOVA.

The results of staining for mice Tg2576 and APP/LxPS1 shown in figure 8B-D and H.

The binding of different antibodies at a concentration of 0.7 μg/ml with the cross-sections of neocortical transgenic mice TG 2576 at the age of 19 months:

C) Parenchymal deposits of Aβ (amyloid plaques) was painted only 6G1 and 6E10, but not selective for globalmenu antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1).

D) for all polling for globalmenu antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1) showed significantly less staining parenchymal plaques compared with the commercially available antibody 6E10 and 4G8.

The binding of different antibodies at a concentration of 0.07-of 7.0 μg/ml with the cross-sections of neocortical transgenic mice APP/LxPS1 at the age of 11 months:

E) Parenchymal deposits of Aβ (amyloid plaques) was significantly stronger painted and at a lower concentration with 6G1, 6E10 and 4G8 than using polling for globalmenu antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1).

All deposits of amyloid were verified through preliminary changefilename staining (Congo red; see Fig. 8A). scale = 100 ám.

Assessment of brown sediment DAB has shown that it is not selective for Aβ antibodies 6G1 and 6E10 painted plaques and vessels of the meninges, while polling for globalmenu Aβ20-42) antibody 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1 not paint. These results showed no binding or significantly less binding of these antibodies to Aβ fibrils or other Aβ species present in amyloid structuresin vivo. Assume that this reduced binding reduces the risk of side effects induced by too rapid dissolution of plaques and the subsequent increase in the level of soluble Aβ or neurospine due to the interaction associated with plaques antibodies with microglia.

The results of staining of the human brain in Alzheimer's disease are shown in figure 8B, F-H.

The binding of different antibodies at a concentration of 0.7 μg/ml with the cross-sections of the neocortex patient RZ55:

B) Parenchymal deposits of Aβ (amyloid plaques) was painted only 6G1 and 6E10, but not selective for globalmenu antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1).

F) For all polling for globalmenu antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1) showed significantly less staining in comparison with the commercially available antibody 6E10 and 4G8.

H) Aβ Deposits in blood vessels (arrows) were stained only 6G1 and 6E10, but not selective for globalmenu antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1).

The binding of different antibodies at a concentration of 0.07-of 7.0 μg/ml with the cross-sections of neocortex the transgenic mice APP/LxPS1 at the age of 11 months:

G) Parenchymal deposits of Aβ (amyloid plaques) was significantly stronger painted and at a lower concentration with 6G1, 6E10 and 4G8 than using polling for globalmenu antibodies (i.e. 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1).

All deposits of amyloid were verified through preliminary changefilename staining (Congo red; see Fig. 8A).

Assessment of brown sediment DAB has shown that it is not selective for Aβ antibodies 6G1 and 6E10 painted plaques and vessels of the meninges, while polling for globalmenu Aβ(20-42) antibodies 5F7, 2F2, 6A2, 4D10, 10F11, 3B10, 7C6, 7E5 and 10C1 not paint. For the commercially available antibody 6E10 and 4G8 showed stronger staining in comparison with the election for globalmenu antibodies, but less staining than 6G1. These results confirm the nature of the staining of the transgenic mice, where there was no binding or attended significantly less binding election for globalmenu antibodies to Aβ fibrils or other Aβ species present in amyloid structuresin vivo. Assume that this reduced binding reduces the risk of side effects induced by too rapid dissolution of plaques and the subsequent increase in the level of soluble Aβ or neurospine due to the interaction associated with plaques antibodies with what microglia.

Example 10: the titer of antibody against Aβ and the profile of selectivity in dot-blotting of plasma TG2576 mice after approximately one year after active immunization.

After approximately one year after the last immunization (globularia Aβ(20-42), globularia Aβ(12-42), the monomer Aβ(1-42) and the media) mice Tg 2576 (from example 9) in the plasma samples was estimated that antibodies against Aβ produced and present. With this purpose, the received serial cultivation of various forms of Aβ(1-42) in the concentration range from 100 pmol/μl to 0.01 pmol/μl in PBS + 0.2 mg/ml BSA. From each sample, 1 μl was applied to a nitrocellulose membrane. Detection was performed using a suitable plasma samples mouse (diluted 1:400). Staining was performed using a conjugate with antibodies against IgG alkaline phosphatase and add coloring reagent NBT/BCIP.

Standards Aβ for dot-blotting:

1. Globular Aβ(1-42)

Getting globular Aβ(1-42) described in example 1a.

2. HFIP pretreated monomer Aβ(1-42) in Pluronic F68

3 mg of Aβ(1-42), (Bachem Inc.; cat. # H-1368) were dissolved in 0.5 ml of HFIP (suspension 6 mg/ml) in 1.7 ml Eppendorff tube and shaked (Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37°C to obtain a clear solution. The sample was dried in the SpeedVac concentrator (1.5 hours) and resuspendable 13.2 μl DMSO, shaken for 10 seconds, with subsequent quenching the rigid ultrasound (20 sec), and shook (for example, in Eppendorff Thermo mixer, 1400 rpm) for 10 minutes was Added 6 ml of 20 mm NaH2PO4; 140 mm NaCl; 0.1% Pluronic F68; pH 7.4 and was stirred for 1 hour at room temperature. The sample was centrifuged for 20 min at 3000g. The supernatant was removed, and the precipitate was dissolved in 0.6 ml of 20 mm NaH2PO4; 140 mm NaCl; 1% Pluronic F68, pH of 7.4. 3.4 ml of H2O was added and was stirred for 1 hour at room temperature followed by centrifugation for 20 min at 3000g. Eight aliquot from each 0.5 ml of the supernatant was kept at -20°C for further use.

3. Globular Aβ(20-42)

Getting globular Aβ(20-42) described in example 1c.

4. Globular Aβ(12-42)

Getting globular Aβ(1-42) described in example 1d.

5. HFIP pretreated monomer Aβ(1-40), 5 mm in DMSO

1 mg of Aβ(1-40), (Bachem Inc., cat. # H-1194) suspended in 0.25 ml of HFIP (suspension 4 mg/ml) in an Eppendorff tube. The tube was shaken (for example, in Eppendorff Thermo mixer, 1400 rpm) for 1.5 h at 37°C to obtain a clear solution and then dried in a SpeedVac concentrator (for 1.5 hours). The sample was re-dissolved in 46 ál DMSO (21,7 mg/ml = 5 mm)was shaken for 10 sec and then treated with ultrasound for 20 sec. After shaking (for example, in Eppendorff Thermo mixer, 1400 rpm) for 10 min, the sample was kept at -20°C for further use is found.

6. The monomer Aβ(1-42), 0.1% of NH4OH

1 mg of Aβ(1-42) (Bachem Inc., cat. # H-1368) were dissolved in 0.5 ml of 0.1% NH4OH in H2O (freshly prepared) (= 2 mg/ml) and immediately shaken for 30 sec at room temperature to obtain a transparent solution. The sample was kept at -20 °C for further use.

7. The fibrils of Aβ(1-42)

1 mg of Aβ(1-42) (Bachem Inc. catalogue Nr.: H-1368) were dissolved in 500 ál of water with 0.1% NH4OH (Eppendorff tube and the sample was stirred for 1 min at room temperature. 100 μl of this freshly prepared solution of Aβ(1-42) was neutralized with 300 μl of 20 mm NaH2PO4; 140 mm NaCl, pH 7,4. the pH was brought to a pH of 7.4 with 1% HCl. The sample was incubated for 24 h at 37°C and centrifuged (10 min at 10000 g). The supernatant was removed and the residue fibrils resuspendable in 400 μl of 20 mm NaH2PO4; 140 mm NaCl, pH 7,4 by shaking for 1 minute

8. sAPPα

Supplied from Sigma (cat. No. S9564; 25 μg in 20 mm NaH2PO4; 140 mm NaCl; pH 7,4). sAPPα were diluted in 20 mm NaH2PO4, 140 mm NaCl, pH of 7.4, 0.2 mg/ml BSA 0.1 mg/ml (= 1 pmol/ál).

Materials for the dot-blot

Standards Aβ:

Serial dilution of Aβ antigens in 20 mm NaH2PO4, 140 mm NaCl, pH 7,4 + 0,2 mg/ml BSA

1) 100 pmol/ál

2) 10 pmol/ál

3) 1 pmol/ál

4) 0.1 pmol/µl

5) 0.01 pmol/ál

Nitrocellulose:

The environment for migration TRANS-blot, net NITR the cellulose membrane (0.45 μm); BIO-RAD

Anti-mouse-AP:

AQ330A (Chemicon)

Reagent for detection:

Tablets NBT/BCIP (Roche)

Bovine serum albumin (BSA):

A-7888 (Fa. SIGMA)

Blocking reagent:

5% low-fat milk in TBS

Buffer solutions:

TBS

25 mm Tris/HCl buffer pH 7.5

+ 150 mm NaCl

TTBS

25 mm Tris/HCl buffer pH 7.5

+ 150 mm NaCl

+ 0,05% Tween 20

PBS + 0.2 mg/ml BSA

20 mm N buffer NaH2PO4a pH of 7.4

+ 140 mm NaCl

+ 0.2 mg/ml BSA

A solution of antibodies I:

Plasma actively immunized TG2576 mice, diluted 1/400 in 20 ml of 1 % milk with a low fat content in TBS

The antibody solution II:

a dilution of 1:5000

Anti-mouse-AP in 1% milk with a low fat content in TBS

The way the dot-blotting:

1) 1 ál each of the different standards Aβ (5 serial dilutions) were marked points on the nitrocellulose membrane at a distance of approximately 1 cm from each other.

2) Points standards Aβ allowed to dry on the nitrocellulose on the air for at least 10 min at room temperature (RT) (= dot-blot)

3) Blocking:

The dot blot was incubated with 30 ml of 5% milk with a low fat content in TBS for 1.5 h at RT.

4) Washing:

The blocking solution was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT.

5) Solution of antibodies I:

The buffer wash was decanted, and the dot blot incubated with a solution of an is the body I over night at RT.

6) Washing:

The solution I antibodies was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The rinse solution was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The rinse solution was decanted and the dot blot incubated under shaking with 20 ml TBS for 10 min at RT.

7) the antibody Solution II:

The buffer wash was decanted, and the dot blot incubated with antibody solution II for 1 hour at RT

8) Washing:

The antibody solution II was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The rinse solution was decanted and the dot blot incubated under shaking with 20 ml TTBS for 10 min at RT. The rinse solution was decanted and the dot blot incubated under shaking with 20 ml TBS for 10 min at RT.

9) the method:

The rinse solution was decanted. 1 tablet NBT/BCIP was dissolved in 20 ml of H2O and the dot blot incubated for 5 min with this solution. The development was stopped by intense washing H2O.

The results are shown in figure 9.

Serum from different groups of immunization: a) globularly Aβ(20-42); b) globularly Aβ(12-42); (c) a monomer Aβ(1-42), 0.1% of NH4OH; and d) control carrier tested against various forms of Aβ in the analysis of the dot-blot testing different profiles of antibodies.

1. Globular Aβ(1-42).

2. The monomer Aβ(1-42), pre-treated the first HFIP, 0.1% Pluronic F68.

3. Globular Aβ(20-42).

4. Globular Aβ(12-42).

5. The monomer Aβ(1-40), HFIP pretreated with 5 mm in DMSO.

6. The monomer Aβ(1-42), dissolved in 0.1% NH4OH.

7. The preparation of fibrils of Aβ(1-42).

8. sAPPα (Sigma); (the first point: 1 pmol).

In contrast, active immunization with a carrier as a control or monomer Aβ(1-42) for immunization globularia Aβ(20-42) or globularia Aβ(12-42) showed high titers of antibodies even after approximately one year after the last immunization. These antibodies are selective for globular Aβ(20-42) in the case of immunization globularia Aβ(20-42) or selective for globular Aβ(20-42) and globular Aβ(12-42) in the case of immunization globularia Aβ(12-42). This indicates that the truncated globular Aβ(20-42) and globular Aβ(12-42) are very good antigens and the antibodies directed against them, have a very long persistencein vivo.

(It should be noted that some of the dot-blots were observed nonspecific staining signal that, most likely, represents a cross-reaction of antibodies of mice BSA used for serial dilutions of peptides Aβ.)

Example 11: Levels of epitope globular Aβ(20-42) in the brain of patients with Alzheimer's disease.

Extract brain with SDS-DTT:

Brain samples from AD: RZ 16; RZ 52 and RZ 55 (obtained from Brain Net, Munich)

One tablet of complete protease inhibitor (Roche, cat. No. 1697 498) was dissolved in 1 ml of H2O (= solution of protease inhibitor). a 100 mg sample of the brain with AD homogenized in 2.5 ml NaH2PO4, 140 mm NaCl, 0,05% Tween 20 and 0.5% BSA (supplemented with 25 μl of a solution of protease inhibitor) with 20 strokes in a glass Potter homogenizer. The suspension was treated with ultrasound for 30 seconds in ice, then incubated at 37°C for 16 hours. The suspension was centrifuged at 100000g and 8°C for one hour, then collected the supernatant. The residue was dissolved in 5 mm NaH2PO4, 35 mm NaCl, pH 7.4 and homogenized with 10 strokes in a glass Potter homogenizer. Added 75 μl of 10% SDS and 125 μl of 0.16 mg/ml DTT and stirred for 20 minutes at ambient temperature. The sample was centrifuged for 10 min at 10000g, and the supernatant was stored over night at -20°C. Before use, the supernatant was thawed and centrifuged for another 10 min at 10000g. The supernatant (= extract brain SDS/DTT) was used for ELISA.

a) Sandwich ELISA for epitope globular Aβ(20-42)

Reagent list:

1. Immunological tablet F96 Cert. Maxisorp NUNC- (cat. No. 439454)

2. Linking antibody: 5F7, 7C6, 10F11

3. The buffer for the binding:

100 mm sodium bicarbonate, pH9,6

4. Blocking reagent for ELISA (Roche Diagnostics GmbH cat. No. 1112589)

5. The PBST buffer:

20 mm NaH2PO4140 mm NaCl, 0,05% Tween 20, pH 7,4

6. Standard Aβ(20-42) for calibration

7. Primary antibody: anti-Aβ pRAb BA199; affinity purified (through globular Aβ(1-42)-sepharose), the IgG solution in PBS; conc.: 0.22 mg/ml

8. Secondary antibody: enzyme conjugate anti-rabbit-POD; (Jackson ImmunoResearch, cat. No. 111-036-045)

9. Development:

- TMB; (Roche Diagnostics GmbH Cat. No. 92817060), 42 mm in DMSO

- 3% H2O2in H2O

- 100 mm sodium acetate, pH 4.9 to

- Stop solution: 2 M sulfuric acid

Obtaining reagents:

1. Linking antibody:

Separate binding antibodies 5F7, 7C6 and 10F11 was diluted to a final concentration of 0.7 μg/ml in buffer for binding.

2. Blocking reagent:

To obtain the source of a blocking solution of blocking reagent was dissolved in 100 ml of H2O and stored at -20°C in the aliquot of 10 ml each.

3 ml of the original blocking solution was diluted with 27 ml of H2O to block one plate for ELISA.

3. Standard Aβ(20-42) for calibration (CS1)

Getting globular Aβ(1-42) described in example 1a. Determined the protein concentration for globular Aβ(20-42) (6.81 in mg/ml by Bradford (BioRad). 14,68 ál globular Aβ(20-42) (6.81 in mg/ml) was diluted in 10 ml of 20 mm NaH2PO4, 140 mm NaCl, 0,05% Tween 20, pH of 7.4, and 0.5% BSA (= 10 μg/ml). 10 μl of a solution of 10 ág/ml was further diluted in 10 ml of 20 mm NaH2PO4, 140 mm NaCl, 0,05% Tween20, pH of 7.4, and 0.5% BSA (= 10 ng/ml = CS1)

Standards for calibration for Aβ(20-42):

Standard for calibrationvolume standard for calibrationPBST + 0.5% of BSAthe final concentration of Aβ(20-42) (PG/ml)CS1.11 ml CS10 ml10000CS1.20,316 ml CS1.10,684 ml3160CS1.30,316 ml CS1.20,684 ml1000CS1.40,316 ml CS1.30,684 ml316CS1.50,316 ml CS1.40,684 ml100CS1.60,316 ml CS1.50,684 ml31,6CS1.70,316 ml CS1.60,684 ml10CS1.80,01.0 ml0,0

extracts m is ZGA with SDS/DTT:

extracts of the brain with SDS/DTT = E#a

(# 4 denotes sample of the human brain (1) RZ 16; (2) RZ 52; (3) RZ 55; (4) RZ 92)

the sample extractthe volume of sample extractPBST + 0.5% of BSAthe dilution factor
E#.11 ml E#0,0 mlwithout cultivation
E#.20,316 ml E#.10,684 ml1:3,16
E#.30,316 ml E#.10,684 ml1:10
E#.40,316 ml E#.10,684 ml1:31,6

4. Primary antibody:

The original solution of anti-Aβ pRAb was diluted to 0.05 μg/ml in PBST + 0.5% of BSA. The antibody solution was used immediately.

5. Secondary antibody:

Lyophilized conjugate anti-rabbit-POD was dissolved in 0.5 ml of H2O and mixed with 500 µl of glycerol. Then concentrate the antibodies were stored at -20°C in the aliquot 100 ál. The concentrate was diluted 1:10000 in PBST buffer. The antibody solution was used immediately.

6. The TMB solution:

20 the l of 100 mm sodium acetate, the pH of 4.9, was mixed with 200 μl of TMB solution and 29.5 μl of 3% hydrogen peroxide. This solution was used immediately.

Tablet for ELISA for Aβ(20-42):

Standards for calibration (CS1.1-CS1.8) and brain extracts in SDS/DTT from 4 samples of the human brain (1) RZ 16; (2) RZ 52; (3) RZ 55; (4) RZ 92 (= E1-E4 4 serial dilutions E#.1-E#.4) was determined in two Parallels:

123456789101112
ACS1.1CS1.1E1.1E1.1E3.1E3.1
BCS1.2CS1.2E1.2E1.2E3.2 E3.2
CCS1.3CS1.3E1.3E1.3E3.3E3.3
DCS1.4CS1.4E1.4E1.4E3.4E3.4
ECS1.5CS1.5E2.1E2.1E4.1E4.1
FCS1.6CS1.6E2.2E2.2E4.2E4.2
GCS1.7CS1.7E2.3E2.3E4.3E4.3
HCS1.8CS1.8E2.4E2.4E4.4E4.4

This ELISA was performed with each of the binding of monoclonal antibodies 5F7, 7C6, 10F11.

Way

1. Add 100 μl of mAb solution per well. Inkubiruut the tablet for ELISA over night at +6°C (refrigerator).

2. Drain the antibody solution and wash wells three times with 250 μl PBST buffer each.

3. Add 250 µl/well of blocking solution. Incubate for 2 hours at ambient temperature.

4. Drain blocking solution and wash the wells three times with 250 μl PBST buffer each.

5. Add 100 ál/well of each standard for calibration and extracts of the brain with SDS/DTT. Incubate the tablet for 2 hours at ambient temperature, then overnight at 6°C.

6. Drain the standards for calibration and solutions of extracts of the brain with SDS/DTT and wash the wells three times with 250 μl PBST buffer each.

7. Add 200 µl/well of a solution of primary antibody and incubate for 1 hour at ambient temperature.

8. Drain the solution of the primary antibody and wash the wells three times with 250 μl PBST buffer each.

9. Add 200 µl/well of a solution of the secondary antibody and incubate for 1 hour at ambient temperature.

10. Drain the solution of the secondary antibody and wash the wells three times with 250 μl PBST buffer each.

11. Add 100 ál/well of TMB solution.

12. To control the color of the tablet during development (5-15 min at ambient temperature) and stop the reaction by adding 50 µl/well of stopping solution in the development of an appropriate color.

13. To measure the absorption at 450 nm.

14. assoicate results using calibration.

15. Evaluation: If the absorption of the samples comes from the linear calibration range, dilute them again and repeat.

The results are shown in figure 10.

Levels of epitope globular Aβ(20-42) in brain extracts of brains from patients with AD and control subjects

Sandwich ELISA was used to evaluate the contents of a truncated epitope globular Aβ(20-42) in extracts of brain. ELISA with the appropriate antibodies against globular Aβ(20-42) used for calibration.

Extraction of brain tissue in Alzheimer's disease showed that the content of the epitope globular Aβ(20-42) significantly increased compared to control patients. This indicates that the epitope of globular Aβ(20-42) is indeed relevant form of Aβ in the brain in Alzheimer's disease, and not only relevant for models of Alzheimer's disease in animals. Antibodies directed against the epitope globular Aβ(20-42), thus, are highly desirable for the treatment of Alzheimer's disease.

Example 12: the Development of cell lines hybridoma against globular Aβ(20-42)

Monoclonal antibodies can be obtained using a wide variety of methods known in this field, including the use of hybridoma, recombinant method and the method of phage display or combinations thereof. For example, monoclonal antibodies can floor is given using hybridoma, including well-known in this field and explain, for example, in Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N. Y., 1981) (the full content of the above links given here in full). The term "monoclonal antibody"as used here is not limited by the antibodies obtained by the method of hybridoma. The term "monoclonal antibody" refers to an antibody derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the way in which it is received.

The specific method used to obtain the antibodies described herein, represent the following:

Immunization of mice: mice Balb/c and A/J (age 6-8 weeks) were immunized subcutaneously with 50 μg of antigen in CFA. Animals were injected booster injections every three weeks with 50 μg of antigen in Immuneasy™ (Qiagen) just three booster injections. Four days before fusion, the mice were administered a booster injection of 10 μg of antigen intravenously.

The cell fusion and screening hybridoma: spleen cells from immunized animals were merged with myeloma cells SP2/0-Ag14 in the ratio of 5:1 using conventional methods. From seven to ten days after the merger, when observed macroscopic colonies, SN were tested by ELISA for antibodies against globular Aβ(20-42). Cells of p is positive on ELISA wells were propagated and cloned by limiting dilution.

Determination of the isotype antibodies: mAb isotypes against globular Aβ(20-42) was determined using kit ittipiboon Zymed EIA.

Getting in large quantities and purification of monoclonal antibodies: hybridoma were propagated in medium containing 5% fetal bovine serum with low IgG content (Hyclone). The supernatant was collected and concentrated. mAb was purified using chromatography with protein A and were dialyzed in PBS.

Titers in serum: Ten mice were immunized with globularia Aβ(20-42). All mice were observed seroconversion with titers in ELISA (1/2 max OD 450 nm) 1:5000-10000.

DENOTE HYBRIDOMAS PRODUCING MONOCLONAL ANTIBODIES

The internal designation of Abbott Laboratories, used for deposition.

The deposited cell line:

1) ML13-7C6.1 D4.4A9.5 G8 (denoted here as well "7C6");

2) ML15-5F7.5B10 (denoted here as well, "5F7");

3) ML15-10F11.3D9 (denoted here as well "10F11");

4) ML15-4B7.3A6 (denoted here as well "4B7");

5) ML15-2F2.3E12 (denoted here as well "2F2");

6) ML15-6A2.4B10 (denoted here as well "6A2");

7) ML13-4D10.3F3 (denoted here as well "4D10");

8) ML15-7E5.5E12 (denoted here as well "7E5");

9) ML15-10C1.5C6.3H4 (denoted here as well "10C1");

10) ML15-3B10.2D5.3F1 (denoted here as well "3B10").

1. Antibody specific for at least one globular β (20-42)containing variable the performance communications region of the heavy (VH) and light (VL) chains, where domains CDR variable regions of the heavy and light chains contain an amino acid sequence selected from the group consisting of the following sets of CDR:

1 set of CDRAmino acid sequence
VH CDR-H1SYGVH
VH CDR-H2VIWRGGRIDYNAAFMS
VH CDR-H3NSDV
VL CDR-L1KSSQSLLDIDGKTYLN
VL CDR-L2LVSKLDS
VL CDR-L3WQGTHFPYT
2 set CDR
VH CDR-H1SYVMH
VH CDR-H2YIYPYNDGTKYNEKFKG
VH CDR-H3TVEGATWDGYFDV
VL CDR-L1KSSQSLLYSKGKTYLN
VL CDR-L2LVSKLDS
VL CDR-L3VQGTHFPHT
3 set CDR
VH CDR-H1SYAMS
VH CDR-H2SIHNRGTIFYLDSVKG
VH CDR-H3GRSNSYAMDY
VL CDR-L1RSTQTLVHRNGDTYLE
VL CDR-L2KVSNRFS
VL CDR-L3FQGSHVPYT
4 set CDR
VH CDR-H1TFYIH
VH CDR-H2MIGPGSGNTYYNEMFKD
VH CDR-H3AKSARAAWFAY
VL CDR-L1RSSQSVVQSNGNTYLE
VL CDR-L2KVSNRFS
VL CDR-L3FQGSHVPPT
5 set CDR
VH CDR-H1TFYIH
VH CDR-H2MIGPGSGNTYYNEMFKD
VH CDR-H3AKSARAAWFAY
VL CDR-L1RSSQSVVQSNGNTYLE
VL CDR-L2KVSNRFS
VL CDR-L3FQGSHVPPT
6 set CDR
VH CDR-H1TFYIH
VH CDR-H2MIGPGSGNTYYNEMFKD
VH CDR-H3AKSHRAAWFAY
VL CDR-L1RSSQSVVQSNGNTYLE
VL CDR-L2KVSNRFF
VL CDR-L3FQGSHVPPT
7 set CDR
VH CDR-H1DYEMV
VH CDR-H2YINSGSGTIHYADTVKG
VH CDR-H3TLLRLHFDY
VL CDR-L1KSSQSLFYSRNQKNFLA
VL CDR-L2WASTGES
VL CDR-L3QQYFSYPWT
8 set CDR
VH CDR-H1DYEMV
VH CDR-H2YISSGSRTIHYADTVKG
VH CDR-H3VL CDR-L1RSSQSLFYRSNQKNFLA
VL CDR-L2WASTRES
VL CDR-L3QQYYSYPWT
9 set CDR
VH CDR-H1DYEMV
VH CDR-H2YISSGSRTIHYADTVKG
VH CDR-H3TLLRLHFDY
VL CDR-L1RSSQSLFYRSNQKNFLA
VL CDR-L2WASTRES
VL CDR-L3QQYYSYPWT
10 set CDR
VH CDR-H1DYVIH
VH CDR-H2YINPYNDGTQYNEKFKG
VH CDR-H3VEGGTWDGYFDV
VL CDR-L1Amino acid sequence of a VL CDR-L1 monoclonal antibodies, which can be obtained from hybridoma outlined in the American type culture collection number escrow MOUTH-7851
VL CDR-L2Amino acid sequence of a VL CDR-L2 monoclonal antibodies which you can get from hybridoma outlined in the American type culture collection number escrow MOUTH-7851
VL CDR-L3Amino acid sequence of a VL CDR-L3 monoclonal antibodies, which can be obtained from hybridoma outlined in the American type culture collection number escrow MOUTH-7851

2. The antibody according to claim 1, where the antibody is humanized.

3. The antibody according to claim 1 or 2, where the antibody contains a human constant region.

4. The antibody according to claim 1 or 2, where the antibody has the character of glycosylation of the person.

5. Antigennegative part of the antibody as defined in any one of claims 1 to 4.

6. Antigennegative part according to claim 5, where the part is a Fab fragment, F(ab')2-a fragment or single-chain Fv fragment of the antibody.

7. The selected nucleic acid encoding the amino acid sequence of the antibody according to any one of claims 1 to 6.

8. Vector to obtain antibodies, where the vector contains the selected nucleic acid according to claim 7.

9. A host cell to obtain antibodies, where a host cell contains a vector of claim 8.

10. A method of producing an antibody comprising culturing the host cell according to claim 9 in culture medium under conditions suitable for the production of antibodies.



 

Same patents:

FIELD: biosynthesis.

SUBSTANCE: invention refers to isolation of nucleic acid molecules that mediate enzymes required for biosynthesis route of lantibiotic 107891 and its homologs. NC contains nucleotide sequence (NS) selected out of a) mlb gene cluster with a certain sequence or b) sequence that corresponds to the sequence a) due to genetic code degeneracy and mediates the same polypeptides, or c) any of ORF NS that mediate polypeptides with the determined amino acid sequence or their combinations. Recombinant vector for transformation of a host cell capable of producing lantibiotic 107891, its homologs and precursors were discovered in the invention. Lantibiotic 107891 or its derivatives are received by culture of host cell transformed by the vector in conditions suitable for cell growth, gene expression and production of the lantibiotic ot its derivative. The isolated polypeptide involved in biosynthesis of lantibiotic 107891 and its homologs is described. Lantibiotic 107891 is beneficial as food supplement and for antibacterial agent preparation.

EFFECT: antimicrobial activity, particularly, against gram-positive bacteria, including methicillin- and vancomycin-resistant strains.

34 cl, 4 dwg, 1 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: recombinant vector of Newcastle disease virus contains an additional transcription unit including a gene coding HA protein of pathogenic avian influenza virus H5 operatively related with a gene start sequence of an initial portion of Newcastle disease virus and a gene end sequence of an end portion of Newcastle disease virus, characterised by the fact that said protein contains an elongation portion consisting of at least three essential amino acids where said elongation portion consists of arginine (Arg) and/or lysine (Lys) residues and contains at least one lysine with said gene not containing a sequence which can be recognised by a viral polymerase of Newcastle disease virus as an end gene sequence.

EFFECT: presented vector enables high expression level of HA protein of pathogenic avian influenza virus H5, as well as vaccine containing such vector provides an immune protection against Newcastle disease virus and avian influenza virus infection.

12 cl, 5 dwg, 1 tbl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, virology and medicine. What is disclosed is a recombinant protein containing one or more polypeptides carrying one or more epitopes of one or more human papilloma virus HPV antigens. Said polypeptides are embedded in one or various permissive sites of adenylatecyclase (CyaA) or its fragments. What is also disclosed is the polypeptide coding such protein and their use in expression systems for producing immunogenic compositions and drugs. The invention can be used in medicine.

EFFECT: CyaA fragment possesses the property of said adenylatecyclase protein for targeted interaction with antigen-presenting cells.

60 cl, 21 dwg, 1 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention describes novel polynucleotide and amino acid sequences of Brachyspira hyodysenteriae, which can be used to diagnose diseases in animals, caused by B. hyodysenteriae, to treat or prevent diseases associated with infection with B. hyodysenteriae. The invention describes a cell containing a plasmid containing a polynucleotide, for treating and preventing a disease associated with infection of an animal with B. Hyodysenteriae. The invention describes immunogenic and vaccine compositions for generating immune response in an animal, which contain a polypeptide, a polynucleotide, a cell or a plasmid for treating or preventing infection of animals by B. hyodysenteriae, as well as sets of instruments for diagnosis which contain a monoclonal antibody, capable of biding the disclosed polypeptide or a polypeptide or polynucleotide. The invention enables to successfully diagnose diseases caused by B. hyodysenteriae, prevent or treat animals infected with B. hyodysenteriae. The sequences described herein can be used for diagnosis and therapeutic and/or preventive treatment of animals from diseases caused by other types of Brachyspira, including B. intermedia, B. suantatina, B. alvinipulli, B. aalborgi, B. innocens, B. murdochii and B. pilosicoli.

EFFECT: high efficiency of using the composition.

39 cl, 4 tbl

FIELD: medicine.

SUBSTANCE: invention relates to versions of glucoamylase with altered properties and to methods of glucoamylase versions application.

EFFECT: reduced formation of condensation products and increase of glucose output during starch conversion.

22 cl, 2 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: recombinant technique is used to obtain a fused polypeptide with activity of interleukin-7, containing a modified human IL-7 molecule in which the T-cell epitope is modified to reduce T-cell response against IL-7, and the Fc part of an immunoglobulin molecule which is fused through its C-end with the N-end of said modified IL-7 molecule. The obtained polypeptide is used in a pharmaceutical composition to stimulate immune response in a patient.

EFFECT: invention enables to obtain a polypeptide with interleukin-7 activity, having low immunising capacity.

8 cl, 43 dwg, 14 tbl, 12 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies specific to CD22 epitope located from amino acid 22 to amino acid 240 CD22. There are disclosed: a coding polynucleotide, an expression vector, a based host cell and a method of producing an antibody with the use of the cell. There are described versions of a method of CD22 detection on the basis of the antibodies. There are disclosed versions of the CD22 immunoconjugate and based pharmaceutical compositions for treating disturbed B-cell proliferation, and also versions of a method of treating with the use of the pharmaceutical composition. There is disclosed a method of B-cell proliferation inhibition on a basis the immunoconjugate. There are described versions of an engineered cystein-substituted antibody specific to CD22 with one or more free cysteines of thiol reactance within the range 0.6 to 1.0. There are disclosed versions of the "antibody-drug" conjugate, the immunoconjugate and pharmaceutical formulaitons for treating disturbed B-cell proliferation. There are also described a method for protein CD22 detection in a sample on the basis of the immunoconjugate, a method for B-cell detection and a method of treating a malignant tumour on the basis of the "antibody-drug" conjugate. There are disclosed: a product for treating disturbed B-cell proliferation on the basis of the pharmaceutical formulation and a method of producing the "antibody-drug" conjugate.

EFFECT: use of the invention provides new specific CD22 antibodies and the based drugs of acceptable therapeutic efficacy with lower toxicity that can find application in therapy of tumours.

227 cl, 25 dwg, 16 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: method involves cultivation of an obligate methanol-assimilating bacterium Methylophilus methylotrophus or Methylobacillus glycogens in a fluid medium with the bacterium secreting an end protein from a bacterial cell where said bacterium has a DNA structure containing a promoter sequence functioning in the methanol-assimilating bacterium, a nucleotide sequence coding a polypeptide containing a signal sequence which functions in the methanol-assimilating bacterium, and a sequence of the end protein functionally connected with the promoter sequence.

EFFECT: method allows producing the protein effectively by means of extracellular secretion, difficult-to-produce by means of secretory production with application of Escherichia coli bacteria.

5 cl, 7 ex

FIELD: medicine.

SUBSTANCE: invention refers to recombinant plasmid DNA pER-Hir coding a hybrid protein capable to autocatalytic breakdown to form [Leul, Thr2]-63-desulphatohirudin, to Escherichia coli to an ER2566/pER-Hir strain - a producer of said protein and a method for producing genetically engineered [Leu 1, Thr2]-63-desulphatohirudin. The presented recombinant plasmid DNA consists of the SapI/BamHI fragment of DNA plasmid pTWIN-1 containing a promoter and a terminator of T7-RNA-polymerase transcription, an amplifier of phages T7 gene 10 translation, β-laktamase (Ap) gene, modified mini-intein Ssp DnaB gene, with an integrated sequence of a chitin-binding domain, and the SapI/BamHI-fragment of DNA containing a sequence of a gene of recombinant [Leul, Thr2]-63-desulphatohirudin-1 containing β-laktamase (Ap) gene as a genetic marker, and unique recognition sites of restriction endonucleases located at the following distance to the left from the site BamHI: Nrul - 186 base pairs, Ndel - 594 base pairs, Xbal - 882 base pairs, EcoRV - 2913 base pairs, Hpal - 2966 base pairs.

EFFECT: inventions allow producing said compound which is used as a drug applied to prevent blood hypercoagulation.

3 cl, 1 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: vector carries an additional transcription unit containing a foreign gene functionally coupled with an overlying starting sequence of a virus gene (GS) of Mononegavirales order and an underlaying end sequence of a virus gene (GE) of Mononegavirales order; between the GS sequence and an initiator codon of the foreign gene and between a stop sign of the foreign gene and the GE sequence there are respectively 3'-noncoding area and 5'-noncoding area (a genome sense chain) of the virus gene of Mononegavirales order.

EFFECT: higher expression level of the protein coded by the foreign gene.

22 cl, 9 dwg, 6 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies specific to CD22 epitope located from amino acid 22 to amino acid 240 CD22. There are disclosed: a coding polynucleotide, an expression vector, a based host cell and a method of producing an antibody with the use of the cell. There are described versions of a method of CD22 detection on the basis of the antibodies. There are disclosed versions of the CD22 immunoconjugate and based pharmaceutical compositions for treating disturbed B-cell proliferation, and also versions of a method of treating with the use of the pharmaceutical composition. There is disclosed a method of B-cell proliferation inhibition on a basis the immunoconjugate. There are described versions of an engineered cystein-substituted antibody specific to CD22 with one or more free cysteines of thiol reactance within the range 0.6 to 1.0. There are disclosed versions of the "antibody-drug" conjugate, the immunoconjugate and pharmaceutical formulaitons for treating disturbed B-cell proliferation. There are also described a method for protein CD22 detection in a sample on the basis of the immunoconjugate, a method for B-cell detection and a method of treating a malignant tumour on the basis of the "antibody-drug" conjugate. There are disclosed: a product for treating disturbed B-cell proliferation on the basis of the pharmaceutical formulation and a method of producing the "antibody-drug" conjugate.

EFFECT: use of the invention provides new specific CD22 antibodies and the based drugs of acceptable therapeutic efficacy with lower toxicity that can find application in therapy of tumours.

227 cl, 25 dwg, 16 tbl, 14 ex

FIELD: medicine.

SUBSTANCE: by recombinant method obtained is fused protein, which contains natural molecule of human erythropoetine with cysteine residue near its C-end and Fc fragment of humal IgG, containing hinge region, N-end of said Fc fragment is connected to said C-end of said erythropoetine molecule, and said Fc fragment is natural, excluding mutation, consisting in substitution of cysteine residue in said hinge region, located the nearest of all to said erythropoetine molecule, with non-cysteine residue, which resulted in the fact that first cysteine residue of said hinge region, located the nearest of all to said N-end, is separated, by, at least, 12 or 17 amino acids from said cysteine residue of said erythropoetine molecule. Obtained peptide is used for stimulation of erythropoesis in mammal.

EFFECT: invention makes it possible to obtain fused protein, which possesses erythropoetine activity, has prolonged time of half-life in vivo in comparison with native human erythropoetine.

43 cl, 20 dwg, 10 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to field of immunology and biotechnology. Claimed are: versions of antibody and antigen-binding fragments of antibody to receptor Il-6 of humans. Considered are: isolated molecule of nucleic acid and vector which contains it. Described are: system "host-vector" and method of obtaining antibody or its antigen-binding fragment, as well as application of antibody or its antigen-binding fragment for obtaining medication.

EFFECT: invention application provides novel antibodies to receptor IL-6 of humans, which can be applied in therapy of IL-6- mediated diseases.

11 cl, 5 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: there are offered: IL-6 receptor antibody, a coding gene, a vector and a host cell for producing the antibody, a method for producing the antibodies, and a pharmaceutical composition for treating IL-6-related diseases containing the antibody.

EFFECT: use of the invention provides new humanised IL-6 receptor antibodies that can find further application in therapy of the IL-6-mediated diseases.

8 cl, 22 dwg, 8 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: substance of the invention involves a humanised human osteopontin antibody containing a variable region of a heavy chain consisting of the amino acid sequence SEQ ID NO:1 and a variable region of a light chain consisting of the amino acid sequence SEQ ID NO:3. Furthermore, the invention involves a polynucleotide containing a sequence coding the variable region of the respective light and heavy chains of the humanised antibody, an expression vector containing polynucleotide, a host cell, a medicine, a method of producing the humanised antibody, a medicine for treating an autoimmune disease, a method of treating, and application of the humanised antibody for producing a pharmaceutical agent.

EFFECT: advantage of the invention consists in creation of the humanised antibody exhibiting improved activity or stability, than activity and stability of standard human osteopontin antibodies.

13 cl, 14 ex, 1 tbl, 16 dwg

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies and their antigen-binding IL-13, particularly human IL-13 specific fragments. There are described: a pharmaceutical composition, a pharmaceutical compound of the antibody, versions of coding and hybridising nucleic acids and expression vectors. There are offered versions of: cells and methods of producing the antibody, methods of treating IL-13 associated disorders. A method of IL-13 detection in a sample is described.

EFFECT: use of the invention provides new IL-13 antibodies with KD about 10-10 M which can be used for diagnosing, preventing or treating one or more IL-13 associated diseases.

87 cl, 37 dwg, 5 tbl, 6 ex

Glypican-3 antibody // 2427588

FIELD: medicine.

SUBSTANCE: versions of antibodies bound with glypican-3 in a site with amino acid residues 1-563 are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: coding polynucleotide, and also a based expression and a host cell on the basis of the vector. There are disclosed: a method of producing the antibody with using a host cell, a cell growth inhibitor on the basis of the antibody, versions of application of the antibody for treating cancer or hepatoma. There is described peptide for producing glypican-3 antibodies containing residues 546-551 of glypican-3. The offered new antibodies exhibit higher cytotoxicity as compared with known glypican-3 antibodies and are specific to a certain site of glypican-3.

EFFECT: invention use can find further application in cancer therapy.

16 cl, 20 dwg, 2 tbl, 27 ex

FIELD: medicine.

SUBSTANCE: what is offered is a human OX40L antibody containing a light chain and a heavy chain each of which contains respectively three CDRs of the light chain and three CDRs of the heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a method of producing and a method of treating with using the antibody.

EFFECT: use of the invention can find further application in therapy of the OX40L mediated immune disorders.

28 cl, 8 dwg, 1 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: versions of the CD38 specific antibodies and their functional fragments are offered. Each version is characterised by the fact that it contains three CDRs of a light chain and three CDRs of a heavy chain. There are described: a coding polynucleotide, and also an expression vector and a host cell including coding polynucleotide. There are disclosed: a pharmaceutical and diagnostic compositions, a method of treating, a method of detecting CD38 in erythrocyte, a method of inducing specific CD38 expressing tumour cell killing with using the antibody. The offered new antibodies exhibit the unexpected properties: to bind minipig's CD38 and to cause cross-linked specific CD38 expressing cell killing.

EFFECT: use of the invention can find further application in therapy of the CD38 mediated disorders.

87 cl, 37 dwg, 4 tbl, 6 ex

Il2 antibodies // 2425054

FIELD: medicine, pharmaceutics.

SUBSTANCE: humanised monoclonal antibody and its active fragment under the invention neutralises human IL2 activity by binding with said human IL2 prior to, during and/or after binding of said human IL2 with human IL2 receptor. A variable light chain region of said antibody contains an adherent amino acid KAPKA sequence in its second frame region, and in addition, in the CDR1-CDR3 regions, contains the amino acid sequences presented in SEQ ID NO 1-3 disclosed in the description, while a variable heavy chain region contains in the CDR1 - CDR3 regions, amino acid sequences presented in SEQ ID NO 4-6 disclosed in the description. The invention describes a polynecleotide molecule coding the antibody under the invention or its active fragment, a pharmaceutical composition based on said antibody or its active fragment exhibiting human IL2 neutralising action, and also an application of said antibody or its active fragment or the polynecleotide molecule coding it for preparing a drug which optionally contains an auxiliary anti-inflammatory or anticancer agent for treating inflammatory diseases or tumours, respectively.

EFFECT: production of the alternative specific IL2 activity inhibitors which directly bind with human IL2.

24 cl, 18 dwg, 4 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: what is offered is an expression construct for expression of single- or multipass transmembrane polypeptides in a bacterial host cell. Said construct contains a protein-coding polynucleotide, a strictly sensitive promoter of lower basal activity in the host-cell, and a leader sequence comprising a translation initiation enhancer. The strictly sensitive promoter comprises at least one positive control element and at least one negative control element. One or more positive and negative control elements represent a heterologous control element. Besides, what is offered is a method for producing the expressed transmembrane polypeptide and a method of recovering it form the host cell.

EFFECT: methods provide producing the high-yield transmembrane polypeptides either of native conformation, or in a soluble form.

53 cl, 28 dwg, 5 tbl, 10 ex

Up!