Conformational anomalous forms of tau proteins and specific antibodies to them
FIELD: immunology, biotechnology.
SUBSTANCE: invention relates to antibodies showing specificity to anomalous processed form of human tau protein that differs by conformation from the normal tau protein and doesn't bind with normal tau protein. Also, invention relates to conformational distinctive tau proteins ("tauones") and diagnostic and therapeutic aspects related to Alzheimer's disease and related taupathies. Proposed antibodies are produced by hybridomas DC-11 or Dc-11/1 deposited in ECACC at numbers 00082215 and 00082216. Also, invention described truncated forms of human tau protein that are truncated by N- and/or C-end and comprise amino acid residues from amino acid 300 to amino acid 400 in the longest isoform of human tau protein (441 amino acids residues). Above mentioned truncated forms of human tau protein can be recognized specifically by antibodies described above. Also, invention describes a method for assay of truncated forms of tau protein in a patient biological sample using a set comprising a proposed antibody and suitable container. Using the proposed invention provides a suitable target for medicinal preparations with early therapeutic effect used in Alzheimer's disease and other taupathies.
EFFECT: valuable medicinal properties of proteins.
11 cl, 15 dwg, 10 ex
The technical field to which the invention relates.
The invention relates to Alzheimer's disease and other tuptim.
The level of technology
Alzheimer's disease (AD) is one of the most common neurodegenerative diseases that are clinically characterized by progressive and irreversible loss of function of recognition and behavioral functions. The disease may be more than 10 years, developing from moderate symptoms to extremely severe manifestations. From BA affecting about 10% of the population aged above 65 years and 20% of the population aged above 80 years. The result of the development of Western society is that the number affected is growing: at the end of 2000, there are already 5 million patients in the U.S. and approximately 18 million people with senile dementia (dementia) in the world. It is assumed that among these, two-thirds of cases, that is 12 million, will be Alzheimer's disease. It is the fourth biggest killer in the Western world after heart disease, cancer, and heart attacks. The number of people with dementia is growing rapidly. In developed countries by 2025 it is expected twofold increase in the number of people with dementia compared with 1980. The cost of caring for the sick to society are huge. For example, the social costs in the United States on dia who the suspects and care, HELL, mainly for custody, currently estimated at 80 billion U.S. dollars annually. Currently, there is neither predictive diagnostic test, or treatment of ad. Disease, therefore, clinically diagnosed after the onset of initial symptoms by eliminating other forms of dementia. The accumulation of classic stamps, senile (neurotic) plaques, neurofibrillary knots (NFU) in the brain of patients with AD, described the Bavarian psychiatrist Aloisa Alzheimer in 1907, remains neuropathologically characteristic of BA.
Common to all of intracellular neurofibrillary structures (neurofibrillary knots, dystrophic neurites and neuropilin thread) are paired helical filaments (PSF). The main protein subunit PSF is associated with microtubule protein Tau, abnormally hyperphosphorylation form (Grundke-Iqbal et al., 1986; Wischik et al., 1988 a, b). Neurons with neurofibrillary changes degenerate, and the degree of degeneration is directly correlated with the degree of dementia in affected individuals (Blessed et al., 1968).
Normal Tau is associated with microtubule protein, which is distributed mainly in axona. Protein Tau is involved in the modulation Assembly, spatial organization and behavior of microtubules (MT)in neurons and, apparently, in the bodies of glial cells (Drewes et al., 1998; Durbin and Kirschner, 1986; Lo Presti et al., 1995). Protein Tau is encoded by a single gene localized on chromosome 17, but is found in multiple isoforms in tissue extracts from adult brain (Goedert et al., 1989; Himmler, A., 1989; Kosik et al., 1989). The heterogeneity of the protein Tau is partly the result of alternative splicing, leading to the formation of up to six isoforms in the brain of adults. These different isoforms differ from each other by the presence or absence of 29 or 58-amino-acid insertions in aminobenzene region, as well as the addition or deletion of tandem repeat (which may be repeated 3 or 4 times) in carboxykinase region Tau, called linking microtubules (MT) domain. This area consists of incomplete repeats of 31 or 32 amino acid residues. In humans, the smallest isoform of Tau contains 352 amino acid residue with three tandem repeats in the MT-binding domain and does not contain even aminobenzoic insert, and the longest isoform contains 441 amino acid residue with four repetitions and both aminobenzene insert. For simplicity, all numbering in the present description refers to the longest isoform of Tau protein man, htau40, containing all inserts (length 441 amino acid residue), according to Goedert et al. (1989).
It is known that many neurologist who ical diseases are characterized by the presence of a filament of cellular inclusions, containing associated with the microtubule protein Tau, namely Alzheimer's disease (ad), progressive supranuclear paralysis (SRP), corticobasal degeneration (CBD), the disease Peak (BP) and a group of related diseases, collectively referred to as frontotemporal dementia syndrome of parkinsonism associated with chromosome 17 (FTDP-17), amiotrophic lateral sclerosis (ALS), a disease of Creutzfeld-Jakob disease (CJD), dementia boxers (DB), a disease of Gerstman-Straussler-Sheinker (BGSS), disease Lewy body (BTL) and Huntington's disease (Dickinson et al., 1998; DiFiglia et al., 1997; Fomo, 1998; Harada and Zimmerman, 1962; Nishimura et al., 1995; Prusiner 1996; Reed et al., 1998; Roberts, 1998; Schmidt et al., 1996; Shankar et al., 1989; Spillantini et al., 1998). Although the etiology, clinical symptoms, pathological parameters and biochemical composition of inclusions in these diseases are different, there is evidence showing that the mechanisms involved in the aggregation of normal cellular proteins in the form of various filament inclusions comparable. It seems that the initial conformation change associated with the microtubule protein Tau, which initiates the formation of nuclei or precursors for the Assembly of the filaments is key. This process may depend on posttranslational modifications of normal proteins, mutations or deletions of certain genes and factors that bind normal the tree and thus, change their conformation. Protein Tau is very hydrophilic. It can be easily extracted from brain tissue or cell culture. For comparison, the filament Tau extracted from brain tissue in Alzheimer's disease, is relatively insoluble. In addition to phosphorylation, normally insoluble and soluble Tau differ according to the degree of post-translational modifications, including glycosylation, lizirovania, Obyedinyonnye and racemization (Kenessey et al., 1995; Ko et al., 1999; Mori et al., 1987; Wang et al., 1996; Yan et al., 1994).
The mechanism by which protein Tau is modified by taking part in the formation of filaments in ad is unknown. Tau is one of the most soluble of the known proteins (Cleveland 1977; a, b; Lee et al., 1988) and therefore it can be aggregated when BA is somewhat mysterious. Fosforilirovanii Tau affect the ability of Tau to form aggregates, causing either stimulatory or inhibitory effects, mainly dependent phosphorylation site (Crowther et al., 1994; Schnider et al., 1999). Numerous in vitro studies have shown that in the presence of a reducing agent, dithiothreitol (DTT), unsaturated free fatty acids, RNA or glycosaminoglycans, normal Tau can be transformed into filaments (Goedert et al., 1996; Kampers et al., 1996; Perez et al., 1996; Wilson and Binder, 1997). Moreover, the formation of filame the TA may also be accelerated in the presence of crosslinking Tau, formed by oxidation on CIS (Schweers et al., 1995). The parameters that were varied in the study of various ways of education filament included the concentration of Tau protein, pH and ionic strength during incubation, many times surpassing existing in the cytoplasm under physiological conditions. In vivo studies of the formed filaments of Tau by scanning transmission electron microscopy (stem) showed that these filaments differ from native paired helical filaments (Ksiezak-Reding, 1998). In the absence of glycans or RNA not detected any PSF-like filaments in samples containing nefosfaurilirovanna or phosphorylated Tau wild type; normal Tau. Research crosslinking-treated heparin Tau, showed that treatment with heparin induces a conformational change of the protein Tau (Paudel and Li, 1999). Collectively, these in vitro agree that: (a) linking microtubules domain is important for the Assembly of Tau filaments; b) the formation of Tau filaments requires conformational (s) change (s) Tau. Simultaneously, these studies show that none of the described modifications of Tau is not able by itself to induce the formation of filament Tau, which correlated with the clinical manifestation of Alzheimer's disease. Identification and description of factors, the mu is necessary to initiate changes in Tau, leading to the formation of the filament in the conditions of the disease, would be important for the development of predictive markers for diagnosis and therapeutic agents that affect the development chaupati.
The aim of the present invention is, thus, providing a suitable target for drugs early therapeutic intervention in Alzheimer's disease and other Touratech. In addition, it is desirable obtaining specific monoclonal antibodies capable of recognizing and interacting with a given target for drugs. This antibody should not only be suitable for predictive determination of molecules, but also for the inhibition and elimination of these molecules, therefore, to be suitable for predictive diagnostics, treatment and prevention of Alzheimer's disease and other chaupati.
These goals relate to the present invention which in one aspect relates to antibodies that are specific to abnormal forms of Tau protein, which are conformationally different from normal Tau, with the indicated antibody is nonspecific to the normal protein Tau. Such abnormal form of the protein Tau, represent a new family of molecules, intra - and extraneuronal localized, soluble and insoluble, site is preferably abnormally protestirovannyx, forms of the protein Tau, which are conformationally different from normal Tau (Novak et al., 1991, 1993). In the present invention it is shown that these conformationally different forms of the protein Tau, called "toonami"according to this specification, are the beginnings, the formation of nuclei for self-developing process of forming filament Tau formations, which correlates with the clinical manifestation of the disease Allgamer, and, thus, tawani are an important therapeutic target in Alzheimer's disease. Tawani of the present invention can be abnormally progressirovanii protein Tau. The biological activity of twonow can be ingibirovany in vitro and within the neurons of the antibodies of the present invention. These antibodies have the ability to detect the presence of Tumanov on predictive stages I, II and III BA, which makes them suitable for predictive diagnosis of this disease. Crucial for the antibodies of the present invention is that only conformationally excellent form of protein Tau (i.e. tauon") recognized by this antibody, whereas normal Tau proteins do not bind to the antibodies of the present invention.
When implementing the present invention excision of modified versions of the forms associated with the microtubule protein Tau in ad would and purified to a homogeneous state and shows they constitute a major part of the filament Tau isolated from neurons affected by Alzheimer's disease. The amino-acid sequence shows that the skeleton of twonow not differ from the skeleton of the protein Tau, but tawani can be distinguished immunologically from normal human Tau due to the different conformation that is detected using conformation-specific monoclonal antibodies of the present invention. Specific examples of such antibodies are monoclonal antibody DC-11, which is produced by the hybridoma cell line stored in the European collection of cell cultures (ACCC) under the Deposit number 00082216, and monoclonal antibody DC-11/I, which is produced by the hybridoma line cells DC-11/I, stored in ECCC Depositary No. 00082215. This family of monoclonal antibodies, which relates to the present invention, is characterized by the recognition of Tawan-specific conformation in the absence of recognition of normal soluble human Tau. Different conformation compared with normal Tau man, was in the pathology inherent in abnormally excision of the modified N-Termini or C-end, or both ends of the molecule Tau, in samples taken in the study in patients with Alzheimer's disease. Interestingly, according to which CNA conformation is not dependent on the isoforms of Tau and the degree of phosphorylation. The essential pathological requirements of Tumanov is to achieve a typical conformation in the presence of Proline-rich and linking microtubules domains and excision of the modified flanking region(s). In addition, Tawana can be distinguished from normal Tau man because of their pathological activity, namely, that Tawana represent beginnings, the centers of formation of nuclei that initiate the aggregation of Tau and Tawana destroy microtubules collected from normal Tau and tubulin. Tawani, pre-incubated with the antibodies of the present invention, in particular monoclonal antibodies family DC-11, showed no ability to the destruction of the aggregates, or collected aggregates of microtubules from normal Tau and tubulin. Moreover, Tawana when microinjections in differentiated neurons and cause significant displacement of endogenous Tau from the faction associated with the microtubule Tau, the retraction of neuronal processes and cell degeneration. If Tawana introduced by microinjection with a monoclonal antibody of the present invention, any of neurodegenerative changes in differentiated neurons was not observed. This shows that the antibodies of the present invention, particularly a monoclonal antibody DC-11, inhibit Akti is the ability of twonow vnutrimatern and therefore can be used as intracellular products (for example, as a therapeutic intracellular antibodies, intracel). Immunohistological, as shown in the example, the antibodies of the present invention, Tawana there are already on-symptomatic stages I, II and III in pre-α-neurons, as in transentorhinal and entorhinal areas with BA, so, after a suitable binding labels, antibodies of the present invention can be used for vital predictive diagnosis of BA.
Preferably, the antibody of the present invention exhibits specificity constituting at least 50%, preferably at least 90% by conformationally great shape Tau ("Tawana"), compared with the antibody DC-11. Specificity can be tested using any of the standard test to determine the specificity of antibodies, such as ELISA, radioimmunoassay, atomic force microscopy and the binding partners, mounted on the console, etc.
In General, all of the antibodies that are specifically reactive against conformationally excellent protein Tau, in particular to its abnormally protestirovanny forms, but not normal soluble Tau, are also included in the scope of the present invention.
Preferably, the antibody of the present invention is specifically reactive" with respect to the molecule, if the about able to contact the molecule by coupling of the molecule with the antibody. The term "epitope" refers to the portion of the antigen that can be recognized and contacted by the antibody. The antigen may have one or more epitopes. "Antigen capable of inducing an animal to produce antibodies capable of binding to the epitope of this antigen. Specific reaction, the above shows that the antigen is highly specific immunoreaction compared to the corresponding antibody, but not to the multitude of other antibodies which can be produced in response to other antigens.
Particularly preferred antibodies of the present invention are antibodies that are derived from a deposited hybridoma cell line DC-11 (ECCC, Depository No. 00082216) and DC-11/I (ECCC, Depository No. 00082215), showing high specificity and selectivity and reactive conformation excellent form of Tau ("Tawana"), but not with normal soluble Tau. Specificity can be determined by any standard available test to determine the specificity of antibodies, such as ELISA, radioimmunodetection etc.
The term "antibody"used herein includes intact molecules and fragments thereof, and synthetic and biological derivatives, such as, for example, fragments, free from Fab, F(ab′)2and Fvor expressed, for example, on the surface of the filament on phage pIII and the and pVIII, or other surface proteins, or on the surface of bacteria, which are capable of binding antigen. The Fab fragments, F(ab′)2and Fvdeprived Fcfragment of intact antibody, more quickly go out of circulation, and may have less non-specific tissue binding of the antibody. In addition, Fvantibody (often referred to as Manantial) can be more easily designed to migrate on With his late specific labels and used for vital early-symptomatic diagnosis of BA, including stage I, II and III BA, not associated with decreased intelligence, which are recognized by the antibodies according to the invention.
In the framework of the present invention, preferred are monoclonal antibodies or fragments of monoclonal antibodies. Thus, according to another aspect, the present invention relates also to the lines of hybridoma cells producing monoclonal antibodies according to the present invention.
The term "Tau", as used in the present description, refers to the long isoforms associate with microtubules human Tau protein, containing all alternative splanirowannya insert, as described M. Goedert et al., 1989.
According to another aspect of the present application, the invention relates to an abnormally protestirovanny form b the LCA Tau, which is conformationally distinct from the protein Tau, and the specified conformationally excellent form of Tau protein is specifically recognized by the antibody of the present invention.
In accordance with this present invention relates to a new family of intra - and extraneuronal localized molecules, soluble and insoluble, abnormally excision of modified forms of the protein Tau, which are conformationally different from normal Tau and are called "toonami".
"Tawana"so, are conformationally perfect forms of the protein Tau, which is specifically recognized by the antibodies of the present invention. Tawani used in the present invention include the sequence, according to PEFC. No. 1, and can be flanked in further amino acids (See. PEFC. No. 2, 3). Tawani contain from 100 to 400 amino acids and is present in this interval excision of modified forms of the protein Tau. Tawani of the present invention can be abnormally excision of modified N - and C-end, or both ends (see Figure 2-13). The term "abnormal excision of modified (abnormally versions)"as it is used here, refers to a peptide Tau ("Tawana")identified in affected neurons in ad, using specific monoclonal antibodies, razrabotannogo the present invention.
Abnormally versions of the form proteins Tau - Tawana - can be obtained using any of the many well-known synthetic recombinant methods. In short, most of the methods that are used to transform cells, construct vectors, extracting messenger RNA, obtaining cDNA libraries and the like, are widely used in the art, and most practitioners are familiar with standard materials which describe specific conditions and procedures. However, for convenience, as a guide can serve the following description.
The most widely used prokaryotic system for production of recombinant proteins in E. coli remains, however, other strains of microorganisms can be used, for example Bacillus subtilis, various species of Pseudomonas, or other strains of bacteria. In such prokaryotic systems are used plasmid vectors which contain replication sites and control sequences derived from species compatible with the host. Commonly used prokaryotic control sequences include promoters for transcription initiation, optionally with an operator, along with sequences containing the binding site of the ribosome.
Now also available with a wide variety eukaryote the definition of the hosts for production of recombinant foreign proteins. Like bacteria, eukaryotic hosts can be transformed systems of expression that directly produce the desired protein, but is more common when provided with a signal sequence affects protein secretion. Eukaryotic systems have an additional advantage because they are able to processional introns that can occur in the genomic sequences coding for proteins of higher organisms. Eukaryotic systems also provide a variety of processing mechanisms that lead to, for example, glycosylation, oxidation or derivatization of certain amino acid residues, conformational control, and so forth.
Commonly used eukaryotic systems include yeast, insect cells, mammalian cells, bird cages and cells of higher plants. This list is not exhaustive. There are suitable promoters that are compatible and operable for use in each of these types of hosts, and terminal sequences and enhancers, such as, for example, the promoter of the baculovirus polyhedron. As mentioned above, the promoters can be costitutivo and doziruemoy. For example, mammalian promoter MTII can be induced by the addition of heavy metal ions.
Professionals in this field are those who Nicky known details of the design of expression systems, suitable for the desired hosts. For recombinant production of the protein encoding DNA properly are ligated into the expression system of choice, and then the system transform into cells compatible host which is then cultured and maintained conditions where the expression of a foreign gene. Tawani of the present invention, obtained in this way is separated from the culture, either by lizirovania cells or from the culture medium, depending on what is appropriate for a specific case and it is known to specialists in this field of technology.
Correct ligation plasmid construction can be confirmed at the initial transformation of suitable host cells in ligiously mixture. Successful transformants are selected by resistance to ampicillin, tetracycline or other antibiotic, or using other markers, depending on the method of constructing plasmids, as is known in the art.
The present invention thus relates to the production of Tumanov, in particular, from the source materials derived from human or from recombinant sources, essentially free from other proteins, in particular from the normal protein Tau. This receipt can be provided by methods that include immunoaffinity stage using ant the bodies of the present invention. Preferably, the preparation according to the present invention contains more than 80% of Tumanov, in particular more than 95% of Tumanov, of the total protein.
Further, the present invention relates also to a kit for determination of Tumanov, abnormally protestirovannyx forms of the protein Tau, which are conformationally different from normal Tau in a sample of brain tissue in Alzheimer's disease or in the sample of body fluid containing the antibody of the present invention, as well as a suitable container for samples. The kit may include antibodies for the identification or selection of Tumanov. Using the antibodies of the present invention sawanoya proteins can be identified and isolated from various sources, including neurons in Alzheimer's disease, transentorhinal and entorhinal areas and hippocampus. Tawani allocated in a similar way, can be further used as immunogen for immunization, for example, mice for constructing a hybrid, producing specific monoclonal antibodies against twonow not recognize normal full-Tau. This method includes the identification and isolation of neurons from transentorhinal and entorhinal areas and the hippocampus from the brain tissue of patients with Alzheimer's disease in a solution that retains the abnormal conformation of Tumanov.
After separation and clear and Tawana used as immunogens and injected subcutaneously to mice at monthly intervals. To construct a hybrid producing monoclonal antibodies against twonow use data spleen of mice. You can get them when using the well known hybridoma techniques first developed by Kohler and Milstein (see .Kohler and .Milstein, "Continuous Cultures of Fused Cell Secreting Antibody of Pre-Defined Specificity", Nature, 256, pp. 495-497, 1975). After quite a long immunization as from the spleen and lymph nodes or peripheral blood of animals get lymphocytes producing antibodies. Preferably, the lymphocytes obtained from the spleen. Then the lymphocytes of the spleen merge with the line of myeloma cells, usually in the presence of contributing to the merge agent, such as polyethylene glycol (PEG). Any of the numerous lines of myeloma cells may be used as a partner to merge in accordance with standard techniques, for example, myeloma line P3-NS1/1-Ag4-1, P3-x63-Ag8.653. Then, the resulting cells, which include the desired hybridoma grow on the selective medium such as HAT, which nalivshiesya cells of the parent myeloma cell or lymphocyte eventually die. Only surviving hybridoma cells, which can be used in certain limiting conditions for obtaining the isolated clones. Supernatant hybrid check for the presence of antibodies of the desired specificity, for example, the R, method of immunological analysis using the antigen used for immunization. Then the positive clones can be subclinical under limiting dilution conditions or on soft agar, and the obtained monoclonal antibodies can be distinguished. Hybridoma obtained using these methods, can be propagated in vitro or in vivo (in ascitic fluid), using methods known in the art. Commonly used cleaning methods monoclonal antibodies include precipitation with ammonium sulfate, ion exchange chromatography and affinity chromatography (see, for example, N. Zola et al., "Techniques for the Production and Characterization of Monoclonal Antibodies," in Monoclonal Hybridoma Antibodies: Techniques and Applications, J.G.R. Hurrel (ed.), pp. 51-52 (CRC Press 1982)).
Further, preferably, the kit of the present invention contains a means for detecting the presence of binding of these antibodies to conformationally excellent protein Tau. Preferred secondary antibody, in particular a secondary antibody, which specifically labeled. In the framework of the present invention can also be used technology using magnetic beads, and other methods of determination of proteins using antibodies. This method includes the determination of Tawana, which is abnormally protestirovanny protein Tau, in the sample to be tested from a patient. The term "subject (TEC the dummy) sample", as used here, refers to the biological sample from the patient, who is suspected in the presence of Tumanov. The test sample may include brain tissue with abnormal versions of the Tau proteins, such as the hippocampal tissue or tissue of the anterior cortex, or the test sample may include cerebrospinal fluid (CSF). In a preferred embodiment of the invention, the test sample includes the CSF, and the identified protein is a CSF-Tawan. The definition of abnormally protestirovannyx protein Tau - Tumanov includes determining in the sample to be tested antigens capable of binding with antibodies specifically reactive against abnormally protestirovanny proteins Tau - tawanan, including the sequence of (Th. No. 1) and flanked by amino acid residues, such as Tawana length in the range of from about 100 to 400 amino acids and is characterized by Tawan-specific conformation that is different from the normal soluble protein Tau, or with antibodies specifically reactive with an abnormally progressirovanii protein Tau - toonami, including the sequence of (Th. No. 1) and flanked by amino acids such as Tawana length in the range of from about 100 to 400 amino acids and is characterized by Tawan-specific conformation that is different from normal plants is Oronogo protein Tau. The presence of Tau protein indicates a disease associated with the accumulation of twonow in patients with BA and other patients with toptime.
A further aspect of the present invention relates to a method for determining abnormal protestirovanny form of the protein Tau, which is conformationally different from normal Tau, in the fluid of the patient's body, comprising mixing a specified body fluids with the antibody of the present invention, the detection of binding between the antibody and conformationally excellent protein Tau (Tawana) and, optionally, measuring the number of conformationally excellent protein Tau, who contacted with the indicated antibody. The presence of Tawana indicates the presence of a disease associated with the accumulation of twonow people, including BA and other chaupati. The body fluid of the patient may present any biological test sample from a person who is suspected in the presence of Tumanov. This fluid body may include brain tissue, such as tissue of the hippocampus or frontal cloth, or cloth of bark, or cerebrospinal fluid (CSF). In a preferred embodiment of the invention tissue fluid includes CSF, and identified protein is a CSF-tuwani.
This definition of twonow can be conveniently supplemented by biochemical and cytochemical methods, or ferment the mi immunological analyses, such as described in many manuals of the manufacturers of kits for immunological assays, as known in the art. If using biochemical methods, it is preferred to use from 0.01 to 10 g, typically from 0.5 to 1 g tissue containing diseased protein Tau, driven to the gel or produce a determination by Western blotting. This method is considered suitable in the absence of age-related controls, which, as shown, are non-reactive to the antibodies of the present invention. Cytochemical methods, staining, as shown, is not reactive with respect to normal tissue.
CSF from patients with ad and patients with neurological diseases that are not related to BA, as well as from healthy subjects were investigated using ELISA for the quantitative determination of Tumanov. The level of twonow in CSF was significantly increased in patients with asthma compared with patients with other neurological diseases, not BA, and controls. When BA was found to be significantly increased, regardless of age genotype of apolipoprotein E and the clinical stage. Western blotting CSF in ad showed several immunoreactive bands with apparent molecular weights between 50 and 15 KD, containing abnormally versions of the protein Tau. These data show that CSF-Tawan which reflect the progressive accumulation of diseased Tau in the development of ad.
In accordance with a further aspect, the antibodies of the present invention can be used to produce drugs for the treatment of patients with Alzheimer's disease. Antibodies can be modified biotechnological methods in molecules with one chain with sequence-target, able to deliver them in neuroblastoma cells expressing tawani. Within this cellular model BA antibodies bind tawani affect their pathological effects (sequestration of normal Tau) and increase the degradation of abnormally protestirovannyx forms of the protein Tau. In vitro studies (sequestration of the protein Tau, the Assembly of the filaments, the destruction of microtubules) with abnormally progressirovanii protein Tau was shown by their correlation with the severity of Alzheimer's disease and shows that they are important targets for drugs.
The present invention will be described in more detail using the following examples and drawings that the invention is not limited to:
figure 1 shows the General scheme of obtaining Tawana;
figure 2 shows a generalized schematic representation of the amino acid sequence of Tumanov;
figure 3 shows the minimum tauon;
figure 4 shows excision modified on the C-end of Tawan;
figure 5 shows poster nslation modified at the N end of Tawan;
6 shows a schematic representation of the protein Tau;
Fig.7 shows Tau 37 person;
Fig shows Tau 39 person;
Fig.9 shows Tau 40 man;
figure 10 shows Tau 43 people;
11 shows Tau 44 people;
Fig shows Tau 46 person;
Fig shows a large Tau rats.
Obtaining monoclonal antibodies family DC 11, specific to Tawana.
To obtain soluble and insoluble of twonow as antigens for immunization (Fig 1)
For selection of twonow from the brain of people with bronchial asthma, have developed a new approach, which is partially based on the methods described Kopke et al. (1993) and Greenberg and Davies (1990). The human brain, with changes, characteristic for stages I-III BA, described Braak, selected short postmortal delay (DMC). Allocated blocks the temporal to the frontal lobe, including interalloy and transentorhinal region, the amygdala and the hippocampus. Tissue was prepared and immediately placed in a minimally sufficient medium (Gibco). The fabric is finely crushed and passed through a sieve with pores of 150 μm. At this stage, the sample of the brain was divided into two aliquots: sample a and sample C.
The sample is then processed in 20 mm TRIS, pH 8, of 0.32 M sucrose, 10 mm β-mercaptoethanol, 5 mm EGTA, 1 mm EDTA, 5 mm MgSO4, 5 mm benzamidine, 10 mm glycerol, 6 mm Fe is iletilenlerin, 50 mm sodium fluoride, 5 μg/ml leupeptin, 1.5 mcg/ml pepstatin and 2 µg/ml Aprotinin and centrifuged at 25000×g for 35 min at 4°With removal of cellular residues. The supernatant was then centrifuged at 200000×g for 40 minutes the precipitate was extracted with 8 M urea at room temperature for 70 min and then centrifuged at 300000×g for 45 min at room temperature. Supernatant were dialyzed for 24 hours against 10 mm TRIS pH to 7.6 with frequent changes, and then were dialyzed for 24 hours against 100 mm MES, 0.5 mm MgCl21 mm EDTA, 2 mm EGTA, 1 mm dithiothreitol, 0.75 mm NaCl, 0.1 mm phenylmethylsulfonyl and 50 mm NaF, pH of 2.7. Saducees proteins were removed by centrifugation at 200000×g for 40 minutes the Supernatant after 200000×g were dialyzed against 25 mm MES, pH 6.4, 0.5 mm MgCl2, 0.1 mm EDTA and 1 mm dithiothreitol and consistently fractionally on the column with phosphate-cellulose which had been equilibrated with the same buffer. The column was loaded with proteins 2 mg/ml and washed with 20 ml linear gradient of NaCl (0-1M) in the equilibrating buffer. Proteins, erwerbende at 0.1-0.8 M NaCl, was assessed by Western blot analysis and were concentrated by speed vacuum apparatus.
The sample was made in 10 volumes of cold buffer (10 mm TRIS, 1 mm EGTA, 0,8 mm NaCl, 10% sucrose, pH 7.4) in a glass homog is nestore. After centrifugation at 27000×g for 30 min at 4°the supernatant was retained, and the residue is homogenized in buffer and centrifuged at 27000×g for 30 min Supernatant after both zentrifugenbau when 27000×g were combined, brought to 1% (weight/volume) N-lauroylsarcosine and 1% (volume/volume) β-mercaptoethanol, and incubated at 37°C for 3 hours on a shaker. After centrifugation at 35000 rpm for 30 min the precipitate homogenized in 5 ml of homogenization buffer, supplemented with 1% mercaptoethanol, and filtered through a 0.45 µm filter. The filtrate was centrifuged at 35000 rpm for 1 hour. Sediment resuspendable in 50 mm Tris, pH 6.8, and was extracted with 2.5% formic acid for 2 min and then centrifuged at 10000×g for 10 min to precipitate insoluble material. Supernatant were dialyzed overnight at 4°against 10 mm Tris, pH 7.4 and centrifuged as before. The obtained supernatant (fraction II) was concentrated using a speed vacuum apparatus, and were determined using SDS-PAGE with subsequent Western blotting analysis. The sediment sample after extraction of 2.5% formic acid containing insoluble Tawana (fraction III)was retained and used for immunization and the dot-analysis. Tawani of fractions (I, II and III) were combined and used as antigens (Figure 1) on the I immunization of mice.
Development of hybridomas producing a family of monoclonal antibodies DC-11.
Balb/c mice six weeks of age were divided into three groups (a, b and C). The first two groups (a, b) initially were immunized with 50 μg of antigen in complete Freund Freud (Sigma) and then again were immunized with 50 μg of the same antigen (AG) in incomplete Freund Freud 5 times with a three-week intervals. In group a all doses were administered in the soles of the feet, and in the group At doses of AG was administered subcutaneously. In the third group of mice was doing the injection of only one dose directly into the spleen in a solution of PBS (vnutristenocna immunization and one week after such premirovany spleen was used for the merge. Three days before fusion, the mice of groups a and b was intravenously injected 50 μg of immunogen in PBS. The spleen cells of immunized mice were used for fusion with cells of the myeloma NS/0 method Konteskova et al., 1988. Splenocytes of 108was mixed with 2×107cell myeloma NS/0 (in the ratio 5:1) and poured in for 1 minute in 1 ml of 50 PEG 1550 (Serva) in a modified environment Dulbecco-Needle (DMEM)without serum, supplemented with 10% dimethylsulfoxide. Fused cells resuspendable in DMEM containing 20% horse serum, L-glutamine (2 mm), gipoksantin (0.1 mm), aminopterin (0,004 mm), thymidine (0,016 mm) and gentamicin (40 U/ml) at a density of 2.5×105to etoc spleen per well in 96-well plates. Cells were incubated for 10 days at 37°and growing hybridoma was selected products antitoon-specific monoclonal antibodies using ELISA method and by immunohistochemistry.
Screening antitoolbar antibodies using ELISA.
Method ELISA was used to identify monoclonal antibodies in supernatant culture hybridomas, which were aimed directly against Tumanov. Used solid-phase tawani, obtained as described above, with the following modifications. Pooled fractions obtained during high-speed vacuum concentration of Tumanov, were separated by electrophoresis on polyacrylamide gel, versions of the forms of Tau protein was isolated by electroelution method Donofrio et al. (1986) and was determined using polyacrylamide gel electrophoresis (SDS-PAGE). Microtiter tablets were coated overnight abnormally modified protein Tau (10 μg/ml, 50 μg/well) at 4°C in PBS. After fixing 1% obesity dry milk to reduce nonspecific binding, the tablets were washed in PBS-0,05% Tween-20 and incubated with 50 μg/well of culture supernatant for 1 hour at 37°C. bound peroxidase monoclonal antibodies were determined using sheep antimisting Ig conjugated to horseradish peroxidase (DAKO). The reaction was carried out with a solution of o-phenylenediacrylate as is ubstrate peroxidase and stopped with 50 ál of 2 M H 2SO4. The absorption was measured at 492 nm on a tablet reader Multiscan MCC/340 ELISA (Labsystems). Value at least twice that of the negative controls were considered positive.
Positive culture was further subclinically on soft agar, according to the procedure Konteskova et al., 1991. Selected subclones were subjected to repeated screening for specific products antitoolbar monoclonal antibodies.
Immunohistochemical screening antitoolbar antibodies.
Monoclonal body identified as positive when antitoolbar ELISA and negative in relation to normal Tau, were re-selected for their specificity to brain tissue of patients with BA, as described below.
The brain of patients with BA, remote autopsy, cut at intervals of 1 cm coronal plate and kept at -20°C. the Blocks of the hippocampus, entorhinal, frontal, occipital and parietal cortex were fixed in 4% buffered formaldehyde solution for more than 4 days. On vibratome cut frontal sections (50 µm) and stored in PBS (pH 7.0) at 4°C. Free-floating on the surface vibratome slices pre-processed within 2-3 min 98% cold formic acid, incubated in preimmune serum in PBS/Triton X 100. Used serum was from the same species as the secondary antibody is. Incubation of slices was performed with a monoclonal antibody positive in the analysis using ELISA method (as described above), for 60 min at 37°C.
Incubation with a second biotinylated antibody (Vectastain Elite kit. Vector) was performed for 1 hour at room temperature. Immunoreactive sites were visualized avidin-Biotin-peroxidase complex (Vectastain Elite kit. Vector) and 6 mg 3-3-diaminobenzidine-4 HCl (SIGMA), 250 mg NiCl2(MERCK) in 10 ml of 0.1 M acetate buffer (pH 6) with 100 μl of N2O2. The reaction was stopped by washing the sections in PBS/Triton (Kiss et al., 1988; Cuello et al., 1993; Thorpe and Kerr, 1994).
Quantitative determination of abnormally protestirovannyx protein Tau (Tumanov) using a family of monoclonal antibodies DC-11
Tawana were isolated as described above. The combination of monoclonal antibodies DC 30 (recognizing both normal and pathological Tau) and a family of monoclonal antibodies DC-11 (specific to abnormally protestirovanny Tau) allows quantification of twonow in the test samples obtained from the brain of patients with AD. Antibodies were isolated from free from serum environment by column chromatography on protein A. microtiter plates (Nunc) with holes with high binding was covered with a mixture of monoclonal antibodies DC-11 at a concentration of 10 μg/ml (50 μl/well) in PBS during the course the e night at 4° C. Nonspecific binding in the wells were saturated by adding 200 μl of 1% skim milk powder in phosphate buffered saline (PBS) for 60 min at room temperature. The tablets were washed 3 times in PBS-0.05 to the Tween-20 (vol./vol.). Added serially diluted standards containing recombinant tawani in concentrations in the range of 100-1000 PG/ml in PBS, and the samples containing BA-tuwani in the amount of 50 µl. After incubation for 60 min at 37°the tablets were washed and added to the antibody DC 30, conjugated with horseradish peroxidase, diluted 1/5000 in PBS (50 μl/well) for 30 minutes at 37°C. After the last wash was added to the wells, 50 μl of a solution of orthophenylphenol and 0.003% H2O2and the plates were incubated in the dark for 20 minutes the Reaction was stopped with 50 ál of 2 M H2SO4. The absorption at 492 nm was read tablet ELISA reader Multiscan MS 344 (Labsystems, Finland).
From the obtained values of the constructed standard curve of recombinant Tumanov and determine the corresponding concentration in the test samples based on the standard curve.
The definition of twonow by Western blotting using monoclonal antibody DC-11
Purified recombinant full-Tau man and abnormally versions of the proteins Tau - tawani were applied to SDS-policr laminae gels with a gradient of 5-20% and dispersed under denaturing conditions, according to Laemmli (1970). After SDS-PAGE produced a transfer for 1 h at polyvinyldifluoride membrane (Millipore) in a buffer of 10 mm CAPS, pH 12, at 350 mA, with cooling. After blotting the membrane was washed in PBS and fixed in 1% dry fat-free milk in PBS for 1 hour at room temperature. Transferred proteins were incubated overnight at 4°With a monoclonal antibody DC-11. After washing in PBS-0.05% Tween-20 (vol./about.) put rat antimachine immunoglobulin, labeled with horseradish peroxidase at a dilution of 1/1000 and incubated 1 hour at room temperature. Then the membrane was washed four times in PBS-Tween-20, showed substrate solution (12 mg 4-chloro-1-naphthol, 4 ml of methanol, 16 PB, 0.03 per cent.about. H2O2), and the reaction was stopped at the H2O. the Results showed that the antibody DC-11 recognizes only abnormally versions of the Tau - tawani; in contrast, monoclonal antibody DC 30, is an antibody for all Tau, recognizing universal all known isoforms of Tau from many species (human, monkey, cow, pig, rat, mouse), regardless of the state of post-translational modifications.
Immunohistochemical identification of twonow
A family of monoclonal antibodies DC-11 is suitable for visualization Tiunov brain at BA at the various types of immunohistochemical procedures.
Tagging to light the howling microscopy
The brain of patients with BA, remote autopsy, was divided into sections with an interval of 1 cm coronal plate and kept at -20°C. the Blocks of the hippocampus, entorhinal, temporal, frontal, occipital and parietal cortex were fixed in 4% buffered formaldehyde solution at 4°C for more than 4 days. A series of frontal sections (50 µm) were cut on vibratome and stored in PBS (pH 7) at 4°C. Free-floating on the surface vibratome slices were treated for 2 min 98% cold formic acid, incubated with preimmune serum in PBS/Triton X 100. Used serum from the same animal species as the secondary antibody. Incubation of sections with a monoclonal antibody DC-11 was carried out for 60 min at 37°C. Incubation with biotinylated secondary antibody (Vectastain Elite kit, Vector) was performed for 1 hour at room temperature. Immunoreactive visualized using the avidin-Biotin-peroxidase complex (Vectastain Elite kit, Vector) and 6 mg 3-3-diaminobenzidine-4 HCl (SIGMA), 250 mg NiCl2(MERCK) in 10 ml of 0.1 M acetate buffer (pH 6) with 100 μl of N2O2. The reaction was stopped by washing the sections in PBS/Triton (Kiss et al., 1988; Cuello et al., 1993, Thorpe and Kerr", 1994).
Double staining for light microscopy
Vibratome slices floating on the surface, were processed within 2-3 min 98% cold is th formic acid, incubated with preimmune serum in PBS/Triton X 100. Used serum from the same animal species as the secondary antibody. Slices were incubated with the first monoclonal antibody DC-11, kongugirovanny with peroxidase at a dilution of 1:1000, in fixing solution (5% horse serum, PBS, 0.1% Triton) for 60 min at 37°showed 0.06% DAB, 0.01% of N2About2in PBS (pH of 7.2). The reaction was stopped by washing the sections in PBS/Triton. Incubation of the same slices with a secondary monoclonal antibody was carried out for 60 min at 37°C. Incubation with biotinylated antibody (Vectastain Elite kit. Vector) was performed for 1 hour at room temperature. The reaction was visualized using the avidin-Biotin-peroxidase complex (Vectastain Elite kit, Vector) and 0.06% 3-3-diaminobenzidine-4 HCl (SIGMA), 0.01% of H2O2, 2,5% mg NiCl2(MERCK) in 0.1 M acetate buffer and was stopped by washing the sections in 0.1 M acetate buffer (Kiss et al., 1988; Cuello, 1993).
Contrast staining quick crazylove purple
After immunohistochemical staining, the sections were placed on glass plates and left in the incubator for 60 min at 56°C. After incubation, the plates were placed in distilled water for 5 min, stained with a solution fast Krasilovka purple for 5-10 min at 4°, washed with water, ZAT is m transferred in 96% ethanol until until the wash is a large part Krasilovka violet, rinsed in xylene and embedded in Entellan.
Vibratome sections (30 μm), freely floating on the surface, were treated for 2 min 98% cold formic acid, incubated with preimmune serum in PBS/Triton X 100. Sections were incubated with primary monoclonal antibody DC-11 for 60 min at 37°C, and then incubated for 30 min with a secondary labeled fluorescent artemisinin antibody goat (Immunotech), diluted 1:500 in PBS/Triton at room temperature, according to standard techniques used in this field. After washing PBS the sections were incubated with labeled TRITS primary antibody for 60 min at 37°and filled With 0.1% solution of paraphenylenediamine on the glycerin.
The Assembly of microtubules and determining binding of microtubules with toonami
The allocation of tubulin
Tubulin was isolated from fresh brain pigs, obtained at a local slaughterhouse, by temperature-dependent cycles of polymerization and depolymerization microtubules, with subsequent ion exchange chromatography (Valee, 1986) phosphocellulose (Watman P11).
The definition of the Assembly of microtubules
Cleaned Tawana (5 mm) was mixed with pre-clarified purified tubulin (10 mm) and GTP (1 mm) in buffer Assembly (100 mm PIPES pH 6.9, 2 mm EGTA, 1 mm MgSO4) at +4°C. This concentration of tubulin below the critical concentration for spontaneous assemblies (Black, 1987). Samples transferred by pipette in a quartz cuvette, preheated to 37°C. the Change in turbidity was determined spectrophotometrically in a thermostatically controlled spectrophotometer (LKB) and recorded changes in OP350with 10-second intervals for 30 minutes
Determination of binding microtubules
Curves of binding of twonow with microtubules was measured as described previously (Gustke, 1992). Purified tubulin were incubated at 37°in the presence of GTP (1 mm) and Taxol (20 μm) in binding buffer (100 mm PIPES pH 6,9,1 mm EGTA, 1 mm MgSO4,1 mm DTT) for 10 minutes Microtubules stabilized by Taxol, which does not affect the binding of twonow or normal proteins Tau and other MAPs, respectively (Valee, 1986; Wallis, 1993), thereby eliminating the effect of the Assembly of microtubules. Tawana was added at a concentration of 2.5 mm, 5 mm, 7.5 mm, 10 mm, 15 mm, 20 mm, respectively. After centrifugation for 35 min at 43000×g, at 37°precipitation resuspendable in P-buffer (50 mm PIPES pH 6.9, 1 mm EGTA, 0.2 mm MgCl2, 5 mm DTT, 500 mm NaCl). Supernatant and precipitates were analyzed by SDS-PFGE gels (Laemmli, 1970), were stained with silver (Bloom, 1987). Gels were scanned on the scanner HPScanJet (Hewlett-Packard) and perform the analysis on the Macintosh computer using the public program NIH image recognition (developed by the National Institute of health, USA and available online at http://rsb.info.nih.gov/nih-image/). The intensity of the bands were transferred to concentration using the method of internal standards and calibration curves.
The preparation of recombinant human twonow
Recombinant excision of modified forms of twonow received using "Erase a Base System (Promega), according to the technical manual. The system is based on the specific cleavage by exonuclease III integrated DNA starting with stickout 5′. The degree of cleavage was homogeneous at a constant temperature. Gene Tau cloned in the vector 17b through the restriction sites Ndel-EcoRI, producing pet/t. To the C-end of the gene was added restriction site kpni restriction sites and three stop codon in all three reading frames downstream of the transcription site kpni restriction sites. The enzyme EcoRI leaves protruding ends 5′, the substrate for Akzo. KnpI leaves 3′ protruding ends that are resistant to splitting Akzo. 1 μg of vector pet/E40 twice digested EcoRI, kpni restriction sites/NEB, ethanol precipitate was diluted in 20 μl of buffer splitting IxExoIII and were digested with 80 units of exoIII at 37°when the speed of the splitting of 450 bases/min After adding ExoIII bore 2.5 ál of the samples with 30-second intervals, one sample in a mixture of 7.5 μl of S1 nuclease, containing 1.5 units of S1-nuclease, on ice. Samples were incubated at room temperature for 30 min to remove any remaining single-stranded x is the frame. For the preparation of blunt ends used DNA polymerase maple. DH5lf-competent cells transformed legatione mixtures of samples. The subclones were subjected to screening by > PST -Xhol restriction and appropriate design sequenced using T7 primer in the vector pet.
Expression, purification and quantification of recombinant twonow
Tawani expressed in E. Coli BL21 (DE3) (Studier, 1986). Single bacterial clones were inoculable in 500 ml LB AMR (LB medium, 100 μg/ml ampicillin). Bacterial cultures were grown on a rotary shaker until such time as their OP600reaches of 0.6-0.8 and then induced by adding IPTG (final concentration 0.4 mm). After 3 hours, the bacterial cells were besieged by centrifugation at 5000 g for 15 min at 4°WITH (SIGMA 6K15, the rotor 12 500), and the precipitated cells were rapidly frozen in liquid nitrogen and kept at -70°C until further use. For preparation of lysate sediment bacteria resuspendable in buffer A (20 mm PIPES pH 6.9, 50 mm NaCl, 1 mm EGTA, 1 mm MgSO4, 2 mm DTT, 0.1 mm PMSF), the cells were destroyed by scoring on ice for 6 min, and cell fragments were removed by centrifugation at 45 000 rpm for 15 min at +2° (rotor TLA-120.2, Bekman Optima TLX). Supernatant filtered through 0.22 μm filters (Millipore) and Tawana immediately purified by ionoo the tion chromatography on a column of phosphocellulose (phosphate cellulose, Whatman P-11). After application of the sample the column was washed with 10 volumes bugarama A. Tawana was suirable 20 ml linear gradient of NaCL (50 mm - 0.5 mm) in buffer A. fractions of 1 ml were collected, and those that contained proteins that were identified on SDS-PAGE. The fractions containing tawani, were combined and were dialyzed against PBS 3×60 min at 4°C. Aliquots after dialysis was dried under vacuum (SpeedVac) and kept at -20°C. Recombinant tawani quantitatively determined by PAGE, using serially diluted bovine serum albumin (BSA) as a standard marker mass. The gel was stained with Kumasi blue, dried, and the intensity of the bands BSA and Tau were determined using Scion Image Beta 3b, Scion Corp.). Build a calibration curve for BSA and used to quantify Tumanov.
The selection of twonow from the brain of people with BA
For selection of twonow from the brain of people with bronchial asthma have developed a new approach, partially based on the methods described Kopke et al. (1993) and Greenber and Davies (1990). Selected men's minds, with changes typically occur BA I-III according to Braak, with a small postmortal delay (DMC). Selected blocks of the temporal lobe, including entorhinal and transentorhinal region, the amygdala and the hippocampus. Tissue was prepared and immediately placed in a minimally sufficient medium (Gibco). The fabric is finely crushed and about what has uskali through a wire sieve with meshes of 150 μm. At this stage, the brain samples were divided into two aliquots: sample a and sample C.
The sample is then processed in 20 mm TRIS, pH 8, of 0.32 M sucrose, 10 mm β-mercaptoethanol, 5 mm EGTA, 1 mm EDTA, 5 mm MgSO4, 5 mm benzamidine, 10 mm glycerol, 6 mm phenylmethylsulfonyl, 50 mm sodium fluoride, 5 μg/ml leupeptin, 1.5 mcg/ml pepstatin and 2 µg/ml Aprotinin and centrifuged at 25000×g for 35 min at 4°With removal of cellular residues. The supernatant was then besieged at 200000×g for 40 minutes Final precipitate was extracted with 8 M urea at room temperature for 70 min and then centrifuged at 300000×g for 45 min at room temperature. Supernatant were dialyzed for 24 hours against 10 mm TRIS pH to 7.6 with frequent changes, and then were dialyzed for 24 hours against 100 mm MES, 0.5 mm MgCl2, 1 mm EDTA, 2 mm EGTA, 1 mm dithiothreitol, 0.75 mm NaCl, 0.1 mm phenylmethylsulfonyl and 50 mm NaF, pH of 2.7. Saducees proteins were removed by centrifugation at 200000×g for 40 minutes the Supernatant after 200000×g were dialyzed against 25 mm MES, pH 6.4, 0.5 mm MgCl2, 0.1 mm EDTA and 1 mm dithiothreitol, and then fractionally on the column with phosphate-cellulose which had been equilibrated with the same buffer. The column was loaded with 2 mg of protein and was suirable 20 ml linear gradient of NaCl (0-1 m) in the equilibrating buffer. the tree, erwerbende at 0.1-0.8 M NaCl, were evaluated by Western blot analysis and were concentrated by speed vacuum apparatus.
The sample was made in 10 volumes of cold buffer (10 mm TRIS, 1 mm EGTA, 0,8 mm NaCl, 10% sucrose, pH 7.4) in a glass homogenizer. After centrifugation at 27000×g for 30 min at 4°the Supernatant was retained, and the residue is homogenized in buffer and centrifuged at 27000×g for 30 min Supernatant after both zentrifugenbau when 27000×g were combined, brought to 1% (weight/volume) N-lauroylsarcosine and 1% (volume/volume) β-mercaptoethanol, and incubated at 37°C for 3 hours with shaking on the shaker. After centrifugation at 35000 rpm for 30 min, the precipitate homogenized in 5 ml of homogenization buffer supplemented with 1% mercaptoethanol, and filtered through a 0.45 µm filter. The filtrate was centrifuged at 35000 rpm for 1 hour. Sediment resuspendable in 50 mm Tris, pH 6.8, and was extracted with 2.5% formic acid for 2 min and then centrifuged at 10000×g for 10 min to precipitate insoluble material. Supernatant were dialyzed overnight at 4°against 10 mm Tris, pH 7.4 and centrifuged as before. The final supernatant was concentrated using a speed vacuum apparatus, and evaluated on SDS-PAGE with subsequent Western blot analysis.
NOTE THE P 8
Purification of normal Tau from brain tissue of humans, pigs and cows
Tau was purified according to the method of Lindwall and Cole., 1984 with modifications. Brain tissue homogenized (1 mg/ml) in 0.1 mm MES, 0.5 mm MgCl2, 1 mm EGTA, 1 M NaCl, pH 6.5) and centrifuged at 100000×g at 4°C for 90 minutes, the Supernatant was brought to 0.5% (vol./about.) 2-mercaptoethanol, heated at 100°C for 5 min and centrifuged at 20000×g at 4°C for 30 minutes This second supernatant was brought to 45% saturation (NH4)2SO4and was centrifuged at 20000×g, as described above, and the final precipitate resuspendable in MES buffer without NaCl. After precipitation with 2.5% (vol./about.) perchloro acid and further centrifugation at 20000×g final supernatant were dialyzed against 5 mm Tris, pH 7.4 overnight at 4°C.
Sequestration and aggregation of normal Tau in the nodes of the filaments under the influence of twonow
An increasing number of normal Tau (5-100 µg/100 µl) was mixed with a fixed number of twonow isolated from fraction I (10 μg/100 μl). The reaction was carried out in a final volume of 100 μl of binding buffer (100 mm MES pH of 7.6, containing 2 mm of EGTA, 2% bovine serum albumin, 0.5 mm MgCl2, 1 μg of Aprotinin, and 20 μg leupeptin). The mixture was left to react for 45 min at room temperature, then put in 150 ál of 80% sucrose the office of binding and centrifuged for 1 h at 100000× g. Top 150 ál was removed, and the remaining voiced to determine the interaction between toonami and normal Tau by radioimmuno-dot-blot analysis. The presence of Tau in the layer of sucrose shows the proportion of released healthy Tau under the action of Tumanov.
Inhibition of degeneration of neurons family monoclonal antibody DC-11
Neuroblastoma cells with growth factor were sown in triplicate in Petri dishes. The first group received only tawani, the second group received Tawana and a mixture of monoclonal antibodies DC-11.
Detection transtechnik of twonow by immunofluorescence assay
The cells were made permeable, soaking in 0.2% Triton X-100, containing 80 mm PIPES, 1 mm MgCl2, 1 mm EGTA, pH 6.6 for 5 min at room temperature. Fixation of cells was made in 2% paraformaldehyde in the same buffer for 15 min on ice. Tawani determined by detecting indirect immunofluorescence assay system for the detection of rhodamine avidin (SIGMA).
The definition of early inhibition of neuronal differentiation
Cells were grown together with factors that induce differentiation. Estimated level of differentiation. The group of cells containing tawani in the absence of antibodies had a significantly impaired ability to differentiate. However, the group treated with a mixture of Tumanov and antic is l, differentiated to levels comparable with cells from the control group treated with unrelated proteins.
1. The antibody with the specificity of truncated forms of the protein Tau man who shortened or N-end, either on the C-end, or both ends, and these shortened form conformationally different from normal Tau protein human, and these shortened forms include amino acid residues, at least, from amino acid residue 300 to amino acid residue 400 of the longest isoform of Tau man, containing the th 441 amino acid residue, but such antibody is not specific for the normal Tau protein and is produced by the deposited hybridoma lines of cells DC-11 or DC-11/I, stored in ESAS numbers 00082215 and 00082216, or has specificity constituting at least 50% to the specified shortened forms of the protein Tau, compared with an antibody produced by the deposited hybridoma lines of cells DC-11 or DC-11/I, stored in ESAS numbers 00082215 and 00082216.
2. The antibody according to claim 1, which has specificity constituting at least 90% to these truncated forms of Tau protein, compared with an antibody produced by the deposited hybridoma lines of cells DC-11 or DC-11/1 stored in ESAS numbers 00082215 and 00082216.
3. Hybridoma cell producing the antibody described in claim 1.
4. Hybridoma cell according to claim 3, which belongs to the line of hybridoma cells DC-11 deposited in ESAS number 00082215.
5. Hybridoma cell according to claim 3, which belongs to the line of hybridoma cells DC-11/1 deposited in ESAS number 00082216.
6. A shortened form of the protein Tau man who shortened at the N-or C-end, or both ends, and these shortened form conformationally different from normal Tau protein human, and these shortened forms include the amino acid is basic residues, at least, from amino acid residue 300 to amino acid residue 400 of the longest isoform of Tau protein human, containing 441 amino acid residue, the shortened form of the protein Tau man have the ability to specifically recognized by an antibody according to claim 1.
7. Kit for determination or selection of the truncated forms of the protein Tau man according to claim 6 in a sample of brain tissue or body fluids, comprising the antibody according to claim 1 and a suitable container for the sample.
8. The kit according to claim 7, characterized in that it further comprises means for determining the presence of binding of the specified antibodies to truncated forms of the protein Tau man, preferably, secondary antibodies, in particular, is specifically labeled secondary antibodies.
9. Set according to any one of claim 7 or 8, characterized in that it further comprises means for quantitative determination of these truncated forms of the protein Tau man, in particular, the standard preparation of these truncated forms of the protein Tau man.
10. The method of determining the shortened forms of the protein Tau man according to claim 6 in brain tissue or fluid of a patient's body, comprising mixing a specified body fluids with the antibody according to claim 1, the detection of binding between the antibody and a truncated form of the protein Tau man, and optionally, a measurement of the amount of the criminal code of Rosenau form of the protein Tau man, contacting the specified antibody.
11. The use of antibodies according to claim 1 for obtaining a medicinal product for the treatment of patients with Alzheimer's disease.
FIELD: medicine, laboratory diagnostics, pediatrics, neonatology.
SUBSTANCE: in neonatals born in mothers with endemic goiter it is necessary to detect the content of CD25+ and HLA-DR+ lymphocytes in peripheral venous blood on the 5th d of their life and at the value of the first parameter being either equal or below 6.9% and the second parameter being either equal or below 16.5% one should predict the onset of infectious-inflammatory diseases in the course of neonatal period.
EFFECT: higher accuracy, sensitivity and specificity of prediction.
3 ex, 1 tbl
FIELD: medicine, analytical immunology.
SUBSTANCE: invention relates to a set of reagents used in quantitative determination of secretory immunoglobulin A (sIgA) that provides assaying status of topical immunity and immunodeficient states in carrying out analysis of serum and secrets of human body. Proposed set comprises a plate with immobilized monoclonal antibodies, five calibrating samples containing 0; 1.0; 5.0; 10.0 or 20.0 mcg of sIgA/ml, conjugate of murine monoclonal antibodies with horse radish peroxidase and a reagent for carrying out the enzymatic reaction. Murine monoclonal antibodies produced by hybrid strain of animal Mus musculus L., № PKKK (P) 676D cultured cells, are immobilized on a carrier. Conjugate comprises murine monoclonal antibodies produced by another strain - Mus musculus L., № PKKK (P) 677D. The advantage of invention involves enhancing sensitivity in assay of sIgA.
EFFECT: improved assay method.
3 tbl, 5 ex
SUBSTANCE: method involves determining availability of antibodies to basic myelin protein at the first and the third week after burn, by applying immunoenzyme analysis approach. The third week values belonging to the range of 0.1-0.19 ODU, vegetovascular dystonia syndrome is predicted to develop. The values being within the 0.2-0.274 ODU limits, disseminated cerebral microsymptoms are to be predicted. The values being within the 0.275-0.3 ODU limits, focal symptom manifestations syndrome is to be predicted.
EFFECT: high accuracy in predicting cerebral disorders severity degree.
SUBSTANCE: method involves determining relative content of CD16+ monocytes in monocytic gate in peripheral venous blood of barren woman. The value being equal to or less than 45%, external genital endometriosis diagnosis is to be set.
EFFECT: high accuracy, specificity and sensitivity of diagnosis method.
FIELD: medicine; microbiology.
SUBSTANCE: method an be used for specific identification and evaluation of potential ability of non-cultivated forms of vibrio cholerae O1 and O139 serous groups to reversion, which vibrios take place in water solutions. Method is based upon preliminary preparation of bacterial suspension of non-cultivated forms of vibrio cholerae O1 and O139 serous groups and bacterial suspension of reference culture of Vibrio Cholerae O1 and O139 serous groups. The working dilution of bacterial suspensions and monoclonal antibodies conjugate is determined and specific monoclonal antibodies are selected for conservative and labile lactose-peptone medium epitopes followed by dot-immune-enzyme analysis or solid phase immune-enzyme analysis reaction. Then appearance of color point or optical density, being higher than 0,2-0,65, is registered and conclusion is made on presence of non-cultivated forms of Vibrio Cholerae O1 and O139 serous groups in sample and their potential reversion ability. Preliminary preparation of non-cultivated forms is performed due to increase in their concentration up to 106-107 mxkl/ml followed by inactivation. For the purpose 20 mkl of 4% sterile gentamicine solution is added to 1 ml of microbe suspension followed by subsequent cooling of samples down to 3-4 deg C during 2 hours. Bacterial suspension of control culture Vibrio Cholerae O1 or Vibrio Cholerae O31 is made from 18-hours culture, grown onto Martin's agar. After that the suspension is used for preparation of bacterial suspension in physiological solution, pH 7,6 till final concentration of 107 mxkl/ml. Method uses set of specific monoclonal antibodies, got to different epitopes of O-antigen of lactose-peptone medium cultures of Vibrio Cholerae O1 and O139 serous groups: 3A9, D11, 3E4, 4H5, D8D6, C3B4, F8G12, F11E5.
EFFECT: ability of getting express-answer at low concentration of tested cells.
5 cl, 2 tbl
FIELD: medicine, laboratory diagnostics.
SUBSTANCE: it is necessary to study saliva due to carrying out immunoenzymatic assay by applying mycobacterial antigen specifically binding with human secretory immunoglobulin A only, and specific monoclonal antibodies directed against human secretory immunoglobulin A. The present method is considered to be of high efficiency, noninvasive, simple in application, of high information value, specificity and sensitivity to tuberculosis.
EFFECT: higher accuracy of diagnostics.
3 ex, 2 tbl
SUBSTANCE: method involves determining concentrations of carcinogens like aniline, toluyline, diethyl alanine, diphenyl and 1-naphtholamine in urine and urodynamic disorders and proliferative urothelium activity from Ki-67 expression degree data. High carcinogen concentrations and urodynamic disorders being detected apart from the fact of tumor removal and unchanged mucosa biopsy, transurethral prostate resection or urinary bladder neck resection is carried out. Next, liquid consumption increase in postoperative period and carcinogen contact reduction are advised. Three months later, control study is carried out. Carcinogen concentration level remaining high and no proliferative urothelium activity reduction being observed on the background of urodynamic disorders, cystectomy is to be repeatedly carried out.
EFFECT: enhanced effectiveness of treatment; high accuracy of diagnosis.
SUBSTANCE: method involves studying superficial lymphocyte receptors in heparinized venous peripheral blood sample with indirect immunofluorescence method using monoclonal antibodies of ICO series with relative CD3+ cells content being below 30%, CD4+ cells content being below 12.5%, and CD95+ cells content being below 7.7%, prognosis is considered to be unfavorable.
EFFECT: high accuracy of prognosis.
SUBSTANCE: method involves determining relative content of CD8+ AND CD16+ lymphocytes in peripheral blood samples taken from 5-7 days old newborns on the treatment background. The value being equal to or greater than 1.3, the treatment is estimated as effective one. The value being less than 1.3, the treatment is estimated as ineffective one.
EFFECT: high accuracy of estimation; in time adjustable treatment course.
SUBSTANCE: method involves determining β-transforming growth factor concentration in mucous membrane of cervical canal. The value being equal to or greater than 240 pcg/ml, severe uterine cervix dysplasia case is to be diagnosed.
EFFECT: high reliability of diagnosis.
SUBSTANCE: claimed method includes injection of vector construct pX-RSVhGH encoding human growth hormone or pX-Ins, encoding human insulin into parenchyma of 8-9-week rabbit males. Transformation effectiveness of gene constructs is determined by immunohystochemical kit Novocastra (Sigma, USA).
EFFECT: method for genetic transformation of rabbit testicle stem cells with increased effectiveness.
3 dwg, 1 ex
FIELD: biotechnology; morphologic investigation of cultivated cells.
SUBSTANCE: primary culture of fibroplasts is subject to influence of electromagnet radiation during 5 minutes continuously with pulse frequency of 73+- Hz and with pulse amplitude of 50V and higher.
EFFECT: improved effectives of growth; higher density of fibroplasts in culture.
FIELD: biotechnology and virology.
SUBSTANCE: strain of hybrid culturing Rattus norvegicus 122H9 cells is disclosed. Said strain is obtained by fusion of rat myeloma cells 210RC.Y3-Ag1.2.3 with rat spleen cells LOU, immunized with purified ectromelia virus of K-1 strain. Said strain is producer of cross-reactive neutralizing monoclonal antibodies against pathogenic for human orthopoxviruses.
EFFECT: vaccines for prophylaxis and therapy of diseases associated with pathogenic for human orthopoxviruses.
2 tbl, 5 ex
FIELD: medicine, immunology, biotechnology.
SUBSTANCE: invention describes antibody raised against osteopontin that inhibits binding integrin and osteopontin or its fragment wherein integrin recognizes a sequence RGD or sequences SVVYGLR, SVAYGLR, SLAYGLR, SVAYGLK and SVAYRLK of osteopontin. Antibody is prepared in using peptide as an antigen comprising amino acid sequence RGDSVVYGLR or relates sequence RGDSVVYGLR. Also, invention proposes a medicinal agent for treatment of autoimmune disease and a method for treatment of autoimmune diseases based on indicated antibody wherein autoimmune disease is chosen from rheumatism, rheumatic arthritis and osteoarthritis. Invention describes using antibody for producing a medicinal preparation used in treatment of autoimmune disease chosen from rheumatism, rheumatic arthritis and osteoarthritis. Using the invention improved arthritic coefficient, delays development of arthritis and reduces legs edema in experimental arthritis in mice and can be used in medicine.
EFFECT: valuable medicinal properties of antibody.
25 cl, 16 dwg, 13 tbl, 20 ex