Agents and methods for measuring polypeptide neurotoxin and its catalytic and proteolytic activities

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented group of inventions refers to medicine. Presented are methods and devices for measuring processed polypeptide neurotoxin in a solution by subtracting the amount of partially processed and unprocessed neurotoxin from an amount of total neurotoxin. Presented are kits that comprise a system of first capture antibody, second capture antibody and detection antibody, media for computing an amount of mature polypeptide neurotoxin and related instructions.

EFFECT: presented group of inventions enables measuring processed neurotoxin in a preparation effectively.

24 cl, 4 dwg, 3 tbl

 

The present invention relates to means for obtaining polypeptides and quality control. In particular, it relates to a method of determining the amount of processioning (active) neurotoxin polypeptide in a solution containing processionary polypeptide neurotoxin and partially processionary or reprezentirovanii polypeptide neurotoxin. In addition, the present invention relates to a device for determining the above quantities and the set is suitable for performing the method in accordance with the present invention.

Clostridium botulinum and Clostridium tetani produce extremely potent neurotoxins, i.e. botulinum toxins (BoNTs) and tetanus toxin (TeNT), respectively. These clostridial neurotoxins (CNTs) bind specifically to neuronal cells and disrupt the release of neurotransmitter. Each toxin is synthesized as an inactive, reprezentirovanii single-chain protein with a molecular mass of about 150 kDa. Post-translational processing involves the formation of disulfide bridges and limited proteolysis (single-stranded gap) through bacterial protease(s). Active neurotoxin consists of two chains, the N-terminal light chain with a mass of about 50 kDa and a heavy chain with a mass of about 100 kDa, linked by disulfide bond. CNTs are structurally and functionally with�coat of the three domains that is, the catalytic light chain, heavy chain, covering translocation domain (N-terminal half) and the receptor-binding domain (C-terminal half), see Krieglstein 1990, Eur J Biochem 188, 39; Krieglstein 1991, Eur J Biochem 202, 41; Krieglstein 1994, J Protein Chem 13, 49. Botulinum neurotoxins are synthesized as molecular complexes containing protein neurotoxin mass of 150 kDa and associated non-toxic proteins. The size of the complex varies depending on the strain and various Clostridial neurotoxin serotypes ranging from 300 kDa, more than 500 kDa and 900 kDa. Non-toxic proteins in these complexes stabilize the neurotoxin and protect it from degradation, see Sil-berstein 2004, Pain Practice 4, S19-S26.

Clostridium botulinum secretes seven antigenically distinct serotypes, referred to as botulinum neurotoxins A-G (BoNT). All serotypes together with related with tetanus neurotoxin (TeNT), secretively Clostridium tetani, are Zn2+-endoprotease that block synaptic exocytosis by cleaving SNARE proteins, see Couesnon, 2006, Microbiology, 152, 759. CNTs cause the flaccid muscular paralysis seen in botulism and tetanus, see Fischer 2007, PNAS 104, 10447.

Despite its toxic effects, the complex of botulinum toxin used as a therapeutic agent for a large number of diseases. Serotype of botulinum toxin And was approved in the United States in 1989 for use in humans to treat strabismus, blepharospasm and other disorders. It is commercially available as a protein preparation of Botulinum toxin A, for example, under the trade name of SATOH (Allergan Inc) or under the trademark DYSPORT (Ipsen Ltd). Improved, free from complexes of the drug Botulinum toxin A, commercially available under the trademark XEOMIN (Merz Pharmaceuticals GmbH). For therapeutic applications, the drug is injected directly into the muscle to be treated. At physiological pH the toxin is released from the protein complex and achieves the desired pharmacological effect. The effect of botulinum toxin is only temporary, which is one reason that repeated administration of botulinum toxin is needed to maintain therapeutic effect.

Clostridial neurotoxins arbitrarily weaken the strength of the twitch muscles and are an effective therapy for strabismus, focal dystonia, including cervical dystonia, and benign essential blepharospasm. In addition, it was shown relief of hemifacial spasm, and focal spasticity, and, moreover, it was shown the ability to be effective under a wide range of other indications, such as gastrointestinal disorders, excessive sweating and cosmetic wrinkle correction, see Jost 2007, Drugs 67, 669.

In x�de process of producing clostridial neurotoxins quantification and quality control of the active neurotoxin polypeptide is particularly important. Currently available drugs contain neurotoxins, in addition to the desired active (protestirovanny or Mature) the neurotoxin proteoliticeski reprezentirovanii predecessor or partly processionary polypeptide neurotoxin. Proteoliticeski reprezentirovanii predecessor or partly processionary the neurotoxin polypeptide differ from the Mature (active, processioning) neurotoxic polypeptide in sequence by only a few amino acids. Therefore, they can hardly be quantitatively distinguishable on the basis of their chemical and physical properties. On the other hand, some proteoliticeski reprocessing predecessor and/or partially processionary polypeptide neurotoxin full of protein can still be of considerable value in such preparations. This part depends on the biological system used for the production, and it results from the biosynthesis and conditions of the fermentation process. Thus, the required number of Mature, biologically active neurotoxin polypeptide in the neurotoxic drugs predefined and currently quite difficult to determine.

Means and the ways of reliable qualitative and quantitative detection of Mature (active) neurotoxin polypeptide is highly desirable, but still insufficient�PNY.

Thus, the technical problem underlying the present invention may be regarded as the provision of means and methods that provide the aforementioned needs. The technical problem is solved by embodiments of the described in the claims and shown below.

The present invention relates to a method of determining the amount of processioning (active) neurotoxin polypeptide in a solution containing processionary polypeptide neurotoxin and partially processionary and/or reprezentirovanii polypeptide neurotoxin containing stages:

(a) contacting a first part of the solution to the first capture antibody that specifically binds to the light chain of a Mature polypeptide neurotoxin, partially processioning and reprocessing of neurotoxin polypeptide, under conditions which ensure the binding of the said antibody with the above-mentioned Mature neurotoxin, partially protestirovanny and reprezentirovanii a polypeptide neurotoxin, with the formation, thus, the first antibody-containing complex,

(b) contacting the first antibody-containing complex with the antibody detection, which specifically binds to the heavy chain of the above-mentioned Mature neurotoxin, partially processor�bath and reprocessing of neurotoxin polypeptide in an antibody-containing complex, formed in step a), whereby is formed a first set of detection,

(c) contacting the second part of the above solution with a second capture antibody that specifically binds to the linkers mentioned partially processioning and reprocessing of neurotoxin polypeptide, under conditions that allow binding of the said antibody with said partially protestirovanny and reprezentirovanii a polypeptide neurotoxin, with the formation, thus, the second antibody-containing complex,

(d) contacting the second antibody-containing complex with the antibody detection, whereby is formed a second set of detection,

(e) determining the amount of the first and second complexes detection formed at the stages b) and d),

f) calculating the amount of Mature neurotoxin polypeptide based on the amounts of the first and second sets of detection determined at the stage (e).

The above method may, in General, contain additional stage comprising a stage for preparation of a solution, or stage relating to additional evaluation results, obtained in stage f). In addition, step a) and b) and stage C) and (d) may be performed simultaneously or sequentially. In the latter case stage a) and b) can be done�us before or after stages C) and d). Further, the definition referred to stage (e) may be held in the said case after both series of stages have been conducted, or the determination at stage (e), as will be discussed the first set of detection is performed after stages a) and b), while the determination regarding the second detection is performed after stages C) and d). The method may be partially or fully automated. The incubation stage and the measurement can be performed, for example, a robot. Data analysis and interpretation can be performed on a computer with the computing algorithm.

The term "neurotoxin polypeptide" used in the present invention, refers to seven individual serotypes of botulinum neurotoxins, i.e. BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, and the tetanus neurotoxin (TeNT), see Table 1, and the options above.

Table 1
Botulinum and tetanus neurotoxins
SEQ ID NO:LinkAccess number:Neurotoxin (full length)/bacterial strain
17Beecher 1997, J Protein Chem 16, 71-712; Krieglstein 1994, J Protein Chem 13, 49-57.ABD65472.1 GI:89258592BoNT/A (Hall/62A)
18Antharavally 1998, J Protein Chem 17, 417-428BAE48264.1 GI:81230332BoNT/B (Okra)
19Sagane 1999, J Protein Chem 18,885-892BAA89713.1 GI:6729213BoNT/CI (C-6814)
20Sagane 1999, J Protein Chem 18,885-892BAA90661.1 GI:6939795BoNT/D (CB16)
21Antharavally 1997, J Protein Chem 16, 787-799CAA43999.1 GI:40394BoNT/E (Beluga)
22Sagane 1999, J Protein Chem 18,885-892CAA73972.1 GI:3805790BoNT/F (NCTC10281)

23Campbell 1993, Biochim. Biophys. Acta 1216 (3), 487-491CAA52275.1 GI:441276BoNT/G
24Krieglstein 1991, Eur J Biochem 202,41-51; Krieglstein et al. 1990, Eur J Biochem 188, 39-45P04958.2 GI:135624TeNT

Neurotoxins mentioned in the present invention, in principle, contain N-terminal light chain and a C-terminal heavy chain. Neurotoxins are produced in the form of single-stranded precursor molecules, referred to in this invention as "reprezentirovanii polypeptide neurotoxins". Sequences N-terminal light chain and the C-terminal the heavy chain is divided into reprezentirovanii the neurotoxins at least one site of proteolytic cleavage. These neurotoxins contain a linker sequence between the sequences of light and heavy chains, where the light chain is localized at the N-Terminus, starting from the first cleavage site, and a heavy chain is localized at the C-end, starting from the second cleavage site. In the aspect of the present invention called the linker has the amino acid sequence as shown in any of the sequences SEQ ID NOs:1-16. During the processing of neurotoxins sequence of the linker will be cut. These neurotoxins contain two sites of proteolytic cleavage, one on the N-terminal and one C-terminal edges of the linker sequence. During the processing of such neurotoxins can occur intermediates, which are cleaved by one or another cleavage site, i.e., the linker sequence is not cut but remains either at the N-limit�th light chain, either the C-terminal of the heavy chain. These intermediates are referred to in the present description as "partially processione polypeptide neurotoxins". Other neurotoxins contain only one cleavage site. It is clear that for these neurotoxins may not be cut any linker sequence. However, reprezentirovanii neurotoxin can be recognized immunologically by the presence of intact site of proteolytic cleavage and flanking sequences. These flanking sequence and the cleavage site are also considered as a linker for the purposes of the present invention. Thus, the term "linker", as used in the present invention and defined above, refers to either the sequence between the sequences of light and heavy chains of polypeptide neurotoxins, which has two cleavage site or the cleavage site and flanking sequences for polypeptide neurotoxins that have only a single cleavage site. As a result of processing obtained "processionary polypeptide neurotoxin". Named neurotoxic polypeptide neurotoxin shows biological properties characteristic of the neurotoxin, namely, (a) binding to a receptor, (b) internalization, (C) translocation through endosomal the membrane in cytosol�, and/or (d) endoproteolytic cleavage of proteins involved in membrane fusion of synaptic vesicles. So processionary the neurotoxin polypeptide is sometimes referred to in the present invention as an active or Mature neurotoxic polypeptide. The biological activity of the polypeptide neurotoxins, in some aspect, is the result of all the above-mentioned biological properties. In vivo studies to assess the biological activity include determining the LD50 for mice and analysis ex vivo of one of the domes of the diaphragm mouse, as described Reise et al. and Dressier et al. (Pearce 1994, Toxicol AppI Pharmacol 128:69-77 and Dressier 2005, Mov Disord 20:1617-1619). Biological activity is usually expressed in mouse units (ME). When used in the present invention, 1 ME - the amount of a neurotoxic component that kills 50% of the treated population of mice after intraperitoneal injection, i.e., LD50for mice by intraperitoneal injection.

In the aspect of the method of the present invention, the aforesaid neurotoxin polypeptide is selected from the group consisting of: (a) neurotoxin polypeptide having the amino acid sequence as shown in any of the sequences SEQ:17-24, and (b) neurotoxin polypeptide having an amino acid sequence which is at least 40% identical to the amino acid sequence �of polypeptide neurotoxin, as shown, any of the sequences SEQ:17-24. The above-mentioned amino acid sequence shown reprezentirovanii polypeptide neurotoxins. The sequence of the corresponding partially protestirovannyx or protestirovannyx polypeptide neurotoxins can be derived from these sequences using the information on the sites of cleavage are presented below in Table 3. In another embodiment of the present invention, the neurotoxin polypeptide has an amino acid sequence which is at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least 99% identical to the amino acid sequence as shown SEQ:17-24. The term "identical", when used in the present invention refers to a sequence identity of amino acid sequences, where sequences are aligned so that achieved the highest degree of compliance. This can be achieved by using published techniques or methods, codified in computer programs, such as, for example, BLASTP, BLASTN, FASTA, Altschul 1990, J Mol Biol 215, 403. The magnitude of percentage identity, in one embodiment, are calculated relative to the complete amino acid sequence. A number of programs based on different algorithms that are available �verifizierung specialist in the art for to compare different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. For conducting sequence alignment, you should use the program PileUp (1987, J Mol Evolution, 25, 351; Higgins 1989 CABIOS 5, 151) or the programs Gap and BestFit (Needleman and Wunsch 1970 J Mol Biol 48; 443; Smith and Waterman 1981, Adv AppI Math 2, 482), which are part of the software package GCG (Genetics Computer Group 1991, 575 Science Drive, Madison, Wisconsin, USA 53711). The values of the identity sequence, the above percentage shall be determined, in one embodiment of the present invention, by using the GAP program in the area of the full sequence with the following settings: weight gap: 50, weight, length: 3, average match: 10.000 and average mismatch: 0.000, which, unless otherwise specified, must always be used as standard settings for sequence alignment. It should be understood that the above-mentioned options, in the aspect of the present invention should maintain at least one of the biological properties of neurotoxins and, in a certain embodiment of the present invention, all of the biological properties of the neurotoxin polypeptide described in the present invention. In the following embodiment of the present invention, the options may be neurotoxins�, having improved or altered biological properties, for example, they may contain cleavage sites which are improved for recognition by the enzyme or to bind to a receptor, or any other property specified above. It is clear that the concept of the present invention is based on the presence of two or more cleavage sites between the light and heavy chain of the neurotoxin polypeptide, while the nature of the cleavage sites and features of amino acid sequence between them does not matter as long as the agent is specific for partially processioning or reprocessing polypeptide neurotoxin. Accordingly, another option is to replace the recognition sites of the protease and the peptide linker between the heavy and light chain of the neurotoxin polypeptide.

In another embodiment, the neurotoxin polypeptide in accordance with the method of the present invention may be a chimeric molecule. The so-called chimeric molecule in one embodiment may have a single substituted domains. Accordingly, in another embodiment, a portion of the heavy chain of the neurotoxin is replaced with the FC-fragment of an antibody.

The term "amount", when used in the method according to the present invention, encompasses the absolute amount of the polypeptide, the relative amount or concentration of the aforementioned polypeptide, as well�, as any value or parameter which correlates with, or can be deduced from them.

The term "solution", when used in the present invention, refers to any solvent system containing Mature neurotoxin polypeptide and partially processione and/or reprezentirovanii predecessors polypeptide neurotoxin. Solvent system further contains a solvent. Possible solvents in various embodiments of the invention are water, aqueous buffers, organic solvents and ionic liquids. In one embodiment of the present invention the solvent system is a solvent system of water. In addition, solvent system in addition to the Mature polypeptide of the neurotoxin and partially protestirovanny or reprezentirovannoe predecessor of the neurotoxin polypeptide and the solvent may also contain additional molecules, including additional bacterial polypeptides. In a certain embodiment, the solution should be used in the method according to the present invention, there will be a culture of bacterial cells or partially purified or purified preparation derived from such a bacterial cell culture.

The term "portion" used in accordance with the method according to the present invention, refers to a sample or to a�iquote solution. In an embodiment of the method of the present invention the first portion and the second portion referred to in the present invention, a fully equivalent in their scope and content. This can be achieved, for example, by measuring the total protein content of the first and second portions, in accordance with which, almost identical total protein is indicative for the first and second portions having substantially the same content. However, in the following embodiment, a portion of which will be used as the first or second portions, may be the dilution of the sample or aliquot of a solution. It should be understood that depending on the amount subject to the determination of the neurotoxin polypeptide (i.e. partially processioning or reprocessing polypeptide neurotoxin or common neurotoxin) dilution may be necessary to ensure optimal quality and quantity. How to make such dilution, is well known to specialists in this field of technology.

The term "contacting" as used in accordance with the method according to the present invention, relates to (i) the provision of the above-mentioned capture antibodies and a solution containing neurotoxins, or (ii) providing an antibody-containing complexes and antibody detection in physical proximity to allow fizicheskoi/or chemical interaction. Suitable conditions which ensure specific interactions, known qualified specialist in this field of technology. These conditions will depend on the antibody and mortar, to be applied in the method according to the present invention, and can be adapted by the person skilled in the art. In addition, the time necessary to carry out the reaction, can also be determined by a skilled person in the art. In addition, you need to understand that between phases in contact, as outlined in the method according to the present invention, can be performed in the washing stage to ensure suitable conditions for contacting. For example, after forming a first antibody-containing complex at the stage a), the remaining solution should be removed before applying the antibodies to detect the indicated antibody-containing complex. In addition, after the first set of detection formed at the stage (b), it may be necessary to remove the remaining (unbound) antibody detection to determine the amount of the first complex detection in stage C). The same applies, of course, for stages from a) to f), respectively.

The term "antibody" as used in the present invention encompasses a monoclonal antibody, polik�first antibody, single-chain antibody, chimeric antibody, bispecific antibody, synthetic antibody, or a fragment of any of the antibodies. Fragments of the aforementioned antibodies include Fab, Fv or scFv fragments, or chemically modified derivatives of any of these fragments. Antibodies may be produced using methods which are described, e.g. in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Monoclonal antibodies can be obtained using the techniques originally described in Kóhler 1975, Nature 256, 495, and Galfre 1981, Meth Enzymol 73, 3. These methods include cell fusion of a mouse myeloma with spleen cells derived from immunized mammals. Antibodies can be further improved using techniques known to specialists in this field of technology. For example, surface plasma resonance, used in the system in BIACORE(R), can be used to increase the efficiency of phage antibodies that bind to the epitope, see Schier 1996, Human Antibodies Hybridomas 7, 97; Malmborg 1995, J. Immunol Methods 183, 7. The antibodies used in the present invention, also contain functional equivalents of antibodies, i.e., agents that are capable of specific binding to desired epitopes or parts of polypeptide neurotoxins. In some embodiment, such functional equivalents include the receptor Il� binding proteins, mentioned in other specified as the specific binding.

According to the method in accordance with the present invention, "the first capture antibody" specifically binds to epitopes contained in the Mature light chain of the neurotoxin polypeptide and partially contained in processional and/or reprezentirovannoe the polypeptide chain of the neurotoxin. The term "specific binding" when used in the present invention, in General, means that the antibody does not cross react substantially with other epitopes on the heavy chain or linker neurotoxic polypeptide to be detected, or on different polypeptides. Specific binding, as mentioned in the present invention, can be investigated by various known methods, including, for example, competition experiments and Western blots. The term "epitope" when used in accordance with the present invention, refers to an antigenic determinant that is recognized by an antibody.

In another embodiment, multiple capture antibody can be used to replace the first antibody capture. This purpose can be used at least one capture antibody that specifically binds to the light chain epitopes reprocessing polypeptide neurotoxin, at least one capture antibody�, which specifically binds to the light chain epitopes partially processioning polypeptide neurotoxin, and at least one capture antibody that is specific binds to the light chain epitopes processioning polypeptide neurotoxin. It should be borne in mind that these three types of antibodies have functional similarity with the first antibody capture for the purposes of the method according to the present invention. Similarly, the capture antibody that specifically binds to the light chain epitopes partially processioning and reprocessing polypeptide neurotoxin, can be used in combination with capture antibody that specifically binds to the light chain epitopes processioning polypeptide neurotoxin.

Specified the first capture antibody in a case must be immobilized. Named immobilization of antibodies, in principle, can be achieved, in some embodiment, reversible or irreversible, direct or indirect (through a linker molecule) binding of an antibody to a solid substrate. In a certain embodiment of the invention, the first capture antibody immobilized to perform method. In another embodiment, the first capture antibody immobilized after was formed first antibody-containing complex, but prior to contacting the complex with a detection antibody�. Materials for solid substrates known to those skilled in the art and include, inter alia, commercially available polysaccharide matrix selected from the group consisting of: separate, Sephadex; agarose, sephacel microcellulose and alginate granules, polypeptide matrices, polystyrene beads, latex granules, magnetic pellets, colloid metal particles, glass, plastic and/or silicon crumbs and surfaces, nitrocellulose strips, membranes, sheets, stabilized red blood cells, wells and walls of reaction plates, plastic tubes. In some embodiment of the present invention, named the solid substrate is made of polystyrene, exposed to gamma radiation.

The term "first antibody-containing complex" refers to a complex containing the first capture antibody that specifically associated with progressirovanii, partially progressirovanii, or is formed by contacting a first capture antibody with a solution containing named processione, partially processione or reprezentirovanii polypeptide neurotoxins, as described above.

According to the method of the present invention, a second capture antibody" specifically binds to an epitope that contains a linker reprocessing and/or partially processioning on�of peptide neurotoxin or its parts. In cases where the linker sequence is absent, provided that the said second capture antibody specifically binds to an epitope containing unsplit the site of proteolytic cleavage. In some aspects of the invention, the second capture antibody does not cross react with protestirovanny a polypeptide neurotoxin to a significant extent. In some embodiment of the present invention, named second immobilized capture antibody specifically binds to the epitope, essentially consisting of the amino acid sequence as shown SEQ ID NO:1-16, containing her or held her, see Tables 2 or 3 below.

Table 2
Amino acid sequence of the cleavage sites of various polypeptide neurotoxins and flanking sequences
SEQThe epitope sequence comprising the cleavage sites (marked)Neurotoxin (bacterial strain)
1KLLCVRGIITSKTKSLDKGYNKALN...DLCIKVBoNT/A (Hall/62A)
2 IQMCKSVKAPG...ICIDVBoNT/B (Okra)
3TKFCHKAIDGRSL...YNKTL...DCRELLVBoNT/CI (C-6814)
4TKVCLRLTK...NSRD...DSTCIKVBoNT/D
5IRFCKNIVSVKG...IRK...SICIEIBoNT/E (Beluga)
6VKFCKSVIPRKG...TKAP...PRLCIRVBoNT/F(NCTC10281)
7IAMCKPVMYKNT GKS......EQCIIVBoNT/G
8IGLCKKIIPPTNIRENLYNRTASLTDLGGELCIKITeNT

CKSVKAPGIC
Table 3
The amino acid sequence of the linker sites
SEQ ID NO:The sequence of epitopesThe sites of cleavageNeurotoxin/bacterial strain
9TKSLDKGYNKK438/T K448/ABoNT/A (Hall/62A)
10K441/ABoNT/B (Okra)
11SLYNKR444/S445 K449/TBoNT/CI (C-6814)
12NSRK442/N443BoNT/D (CB16)

R445/D446
13GIRK419/G420 R422/K423BoNT/E (Beluga)
14KGTKR435/K436 K439/ABoNT/F(NCTC 10281)
15NGTKBoNT/G
16ENLYNRR449 (alternative R455)TeNT

Due to the presence of the above-mentioned epitope, reprezentirovanii or partially processione polypeptide neurotoxins can specifically contact the second capture antibody and, thus, to form a second anti�ate-containing complex. Named second capture antibody is immobilized, as explained in detail above.

Accordingly, the term "second antibody-containing complex" refers to a complex containing a second capture antibody that specifically associated with partially protestirovanny or reprezentirovanii the neurotoxin polypeptide. Named second antibody-containing complex, however, should not contain processionary polypeptide neurotoxin.

According to the method of the present invention "antibody detection" specifically binds with the first and/or second antibody-containing complex. In a certain embodiment of the invention, antibody detection is identical for the first and second antibody-containing complex. However, in another embodiment, the first and second antibody-containing complex can be utilized in a variety of antibody detection. In a certain embodiment, the antibody specifically binds to the detection of the epitopes on the heavy chain processioning, partially processioning and reprocessing polypeptide neurotoxin. Due to the presence of the same epitope in both complexes, the first antibody-containing complex or a second antibody-containing complex can be specifically related, and, thus, detected by antibody detection in the specified embodiment, the present�status (active or inactive of the invention.

As a result of specific binding of antibody detection, are formed a first set of detection or the second set of detection, respectively.

Therefore, the term "first set of detection" refers to a complex containing the first antibody-containing complex and antibody detection. Similarly, the term "second set of detection" refers to a complex containing the second antibody-containing complex and antibody detection.

In the aspect of the method according to the present invention, the specified antibody detection contained in the first or second complex detection, connected with apparently detected the mark, allowing to measure the amount of antibody detection, which is associated with complex detection. By measuring the number of named linked antibody detection may be determined by the number of first or second antibody-containing complexes, as the number of bound antibodies detected in the complex detection correlates with the amount of antibody-containing complex contained in the complex detection. Labeling may be carried out by direct or indirect methods. Direct labeling involves an immediate accession label (covalently or ecovalence) to the first antibody detection. Indirect labeling involves the binding (covalently and�and ecovalence) agent, which specifically binds to the antibody discovery and which carries detectivea label. This agent may be, for example, secondary (higher order) an antibody that specifically binds to the antibody detection. The secondary antibody in this case will be connected with apparently detected the tag. It should be borne in mind that additional antibodies of higher order are also often used for the detection of complex detection. Antibodies of higher order are often used to amplify the signal. Suitable antibodies of higher order can also include well-known system-streptavidin-Biotin (Vector Laboratories, Inc.) and well known Dako LSAB™2 and LSAB™+ (labeled streptavidin-Biotin), or Dako PAP (peroxidase antiperoxidase). In the next version of the specified label of the first antibody detection is selected from the group consisting of: fluorescent dyes, chemiluminescent molecules, radioactive labels, and enzymes capable of generating a detected signal. Typical fluorescent labels include fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein and Texas Red (e.g. Alexa 568). Typical radioactive labels include35S,125I,32P,33P Alternative, etc., detectable label, connected with said first antibody is detected�I, may also be an enzyme that is capable of generating the detected signal, for example, by conversion of the substrate. In some embodiment, the enzyme may be a peroxidase (e.g., horseradish peroxidase) or alkaline phosphatase.

The term "determining the amount" used in the present invention relates to the measurement of absolute amount, relative amount or concentration of the quantitative or semi-quantitative way. The measurement will be carried out on the basis of chemical, physical or biological properties apparently detected the label connected to the first antibody detection. Suitable units of measurement to detect well known to those skilled in the art and depend on the nature of the label apparently detected, as indicated above. However, it should be understood that the number of apparently detected tags, which can be measured, directly correlates with the amount of complex detection, which in turn correlates with the amount of antibody-containing complex and, thus, the amounts defining the type of neurotoxin, which must be defined, either General (processioning, reprocessing and partially processioning neurotoxin) or reprocessing and partially processioning neurotoxin. You should understand the�, the determination of the number of polypeptide neurotoxins, in a variant, also require calibration method using standard solutions with a predetermined amount of the polypeptide neurotoxins. How to perform such calibration is well known to specialists in this field of technology.

The term "calculation" is used in accordance with the method according to the present invention, applies mathematical operations to determine the number processioning neurotoxin based on the total amounts of the neurotoxin (i.e. processioning, reprocessing and partially processioning neurotoxin) and the number of partially processioning and reprocessing neurotoxin. In relation to the method according to the present invention, the above calculations include the subtraction of the number of partially processioning and reprocessing neurotoxin from the total amount of neurotoxin.

Method in accordance with the present invention preferably allows to reliably determine the number processioning neurotoxin in this drug. Accordingly, the quality of drugs on the basis of the neurotoxin can be increased because the drugs can be tested on the constant maintenance required processioning polypeptide neurotoxin.

In Prince�pyo, a method according to the present invention may be formed by attaching a first capture antibody to a solid support, such as the reaction tube. Similarly, the second capture antibody must be connected to another physically separate solid substrate (e.g., with additional reaction vial). Both the capture antibody attached to a solid substrate, will consistently involve contact with said portions of a solution containing processionary, reprezentirovanii and/or partially processionary neurotoxin, to be determined. This solution may be, for example, purified bacterial cell culture Clostridum sp. It should be borne in mind that the first portion will be involved in contact with the first antibody capture on the first solid substrate and the second portion will involve contact with a second capture antibody on a second solid support. The portions are typically of equal volume and normalized with respect to their content, for example, the total protein content. The contacting performed for a time sufficient for specific binding of the first and second capture antibodies with their respective antigens. For example, the contacting can be performed at room temperature for about one hour. Subsequently, the first and second portions of the solution are removed,and the solid substrate (for example, reaction tubes) will be washed once or twice with buffer, under conditions that do not affect the first and second antibody-containing complexes, which were at that time formed with the capture antibodies on the solid substrates. After the washing stage were performed, (first) antibody detection should be added to solid substrates, under conditions that allow specific binding of the antibody detection. Excess antibody detection should be removed further stages of washing with a suitable buffer. Subsequently, the number of the first and second sets of detection can be determined by determining the amount of specifically bound antibody detection. This is achieved depending on the nature of the label antibody detection, for example, by measuring optical density or fluorescence intensity. The measured quantity for apparently detected markers can be compared to the calibration standards to determine the amount of neurotoxin, that is either General (processionary, reprezentirovanii and partially processionary neurotoxin) or reprezentirovanii and partially processionary neurotoxin in the first or second complex detection. It should be borne in mind that the first set of detection represents the total number neurotox�on, while the second set of detection represents the number of only partially protestirovannyx and reprezentirovanii neurotoxin polypeptides. Accordingly, the number processioning polypeptide neurotoxin can be calculated by the aforementioned method, the subtraction amount is partially processioning or reprocessing of the neurotoxin polypeptide of the total amount of neurotoxin polypeptide.

It should be understood that the above definitions and explanations of terms shall apply, with amendments and reservations in respect of all options described in this description, unless otherwise indicated.

The present invention also relates to a method of determining the amount of processioning (active) neurotoxin polypeptide in a solution containing processionary polypeptide neurotoxin and partially processionary and/or reprezentirovanii polypeptide neurotoxin containing stages:

(a) contacting the first portion of the specified solution with a first capture antibody that specifically binds to heavy chains of the Mature polypeptide neurotoxin, partially processioning and reprocessing of neurotoxin polypeptide, under conditions which ensure the binding of these antibodies with the specified Mature neurotoxin, partially protestirovanny and nepriestarauja�tion neurotoxin polypeptide, with the formation, thus, the first antibody-containing complex,

(b) contacting the first antibody-containing complex with the antibody detection, which specifically binds to the light chain of the specified Mature neurotoxin, partially processrunner and reprocessing of neurotoxin polypeptide in an antibody-containing complex formed in step a), whereby is formed a first set of detection,

(c) contacting the second portion of the specified solution with a second capture antibody that specifically binds to the linkers mentioned partially processioning and reprocessing of neurotoxin polypeptide, under conditions which ensure the binding of the indicated antibodies with partially named protestirovanny and reprezentirovanii a polypeptide neurotoxin, with the formation, thus, the second antibody-containing complex,

(d) contacting the second antibody-containing complex with the antibody detection, whereby is formed a second set of detection,

(e) determining the amount of the first and second complexes detection, formed at the stage b) and stage (d), and

f) calculating the amount of Mature neurotoxin polypeptide based on the amounts of the first and second sets of detection determined at the stage (e).

In another embodiment, the methods according to the present invention, these methods also include determining the binding activity of a polypeptide neurotoxin.

The term "binding activity", applied in accordance with the method of the present invention, refers to the ability processioning polypeptide neurotoxin contact surface receptor protein which is present, for example, on peripheral cholinergic nerve endings. Receptor proteins include SV2 for BoNT/A, sinap-totalmini I and II for VAT/b and BoNT/G, and gangliosides (GT1B) co-receptor. In respect of the method according to the present invention, called binding activity may be determined by the method of ex vivo using the model substrate, which replaces the surface receptor protein by simulating its binding domain. Named model the substrate is, in a certain embodiment, the labeled peptide derived from the above-mentioned receptor protein. In another embodiment, suitable markers include those that are listed in another part of the description of the present invention, and in particular Biotin.

Thus, the present invention also relates to a method for determining the binding activity of a polypeptide neurotoxin that contains the stage

(a) contacting portions races�thief containing the neurotoxin polypeptide with a labeled peptide, whereby is formed a complex, and

(b) definition of the specified complex formed in step (a) based on the label, whereby the presence or absence of the complex, or the number indicate the binding activity of a polypeptide neurotoxin in the specified solution.

The complex can be determined on the basis of the nature of the label used for labeling of the peptide. In some embodiment, for example, biotinylating peptide contained in the complex can be determined using streptavidine conjugate capable of generating a detected signal. The presence, absence or intensity would indicate the binding activity of polypeptide neurotoxins in solution or their strength.

In another embodiment of the method according to the present invention, the method further comprises determining proteolytic activity of the polypeptide neurotoxin.

The term "proteolytic activity", applied in accordance with the method of the present invention, relates to the ability processioning neurotoxin proteoliticeski break down sensitive to N-ethylmaleimide factor ensuring the attachment of proteins (SNARE) involved in the fusion of synaptic membrane p�sirica. In a certain embodiment, the specified splitting is zinc-independent. Called proteolytic activity can be determined using the model substrate, which replaces the natural SNARE-protein. Moreover, when splitting a detectable label such as a dye to be released from the named model of the substrate. In one embodiment, a model substrate is a compound having the General formula X-pair-nitroanilide, where X is arginine or a peptide having the sequence arginine-Y in which Y represents one or more amino acids, and in another embodiment, the compound is an arginine-para-nitroanilide.

Thus, the present invention, furthermore, relates to a method for determining the proteolytic activity of the neurotoxin, which includes stages

(a) contacting portions of the solution containing the neurotoxin polypeptide, with a compound having the General formula: X-pair-nitroanilide, where X is arginine or a peptide having the sequence arginine-Y, where Y represents one or more amino acids, and

(b) determining the proteolytic activity of the neurotoxin polypeptide in the specified solution based on the number of released para-nitroaniline at the stage b), which correlates with the amount of neurotoxin polypeptide.

In this embodiment, the Y made�ing the remainder of the peptide, having the amino acid sequence as shown in any of the sequences SEQ ID NOs:25 or 26.

Processionary polypeptide neurotoxin contained the specified portion of the solution may break down and, thus, releasing para-nitroaniline from the remaining peptide. Para-nitroaniline is a dye, well known to those skilled in the art. Determination of proteolytic activity of the neurotoxin polypeptide in the specified solution is based on the number of the released para-nitroaniline, which correlates with the amount of neurotoxin polypeptide.

The present invention also relates to an apparatus for determining the amount processioning of neurotoxin polypeptide in a solution containing:

a) the system first capture antibody, the second antibody capture and antibody detection, where the system allows to carry out stage (a) to (e) above; and

(b) means for calculating the amount of Mature neurotoxin polypeptide based on the amounts of the first and second sets of detection, defined using the system in accordance with a).

The term "device" used in the present invention refers to systems containing at least the above installation and means, operationally associated with one another to do in�zmeinym definition. In some embodiment, the system may be a solid substrate with immobilized capture antibodies, as mentioned above, which can be in physically separated tubes to allow contacting of the first and second portion of the solution. Moreover, the device may comprise a case, a unit for determining the number of complexes detected. Depending on the type of antibody detection, this unit will contain a detector for signals generated by antibody detection. In addition, the block may also contain, in a certain embodiment, the means for calibration, for example, the algorithm based on the computer to compare the measured signals with calibration standards to determine the number of polypeptide neurotoxins present in solution or in portions. The device will also contain a means for calculating the amount of Mature neurotoxin polypeptide based on the number of the first and second sets of detection, for example, a computer algorithm for performing the computation.

In addition, the present invention relates to a kit suitable for carrying out the aforementioned methods, wherein the specified set contains:

a) system of the first capture antibody, the second antibody capture and antibody detection, where the uke�data the system allows carrying out stages a) to e) above ways;

(b) means for calculating the amount of Mature neurotoxin polypeptide based on the amounts of the first and second sets of detection, defined using the system in accordance with a); and

(C) instructions for carrying out this method.

The term "set" used in the present invention, refers to a set of the above means or reagents in accordance with the present invention, which may be packaged together or separately. The components of the kit may contain separate vials (i.e. as a set of separate parts) or be supplied as a single vial. In addition, it should be understood that the kit in accordance with the present invention is designed to implement the methods mentioned in the description of the present invention described above. In one embodiment it is envisaged that all components are delivered in a condition ready for carrying out the above methods. In another embodiment, the kit contains instructions for performing these methods. The instructions can be presented in the form of a guide for the user in paper or electronic form. For example, management may include instructions for interpreting the results obtained during the implementation of the above ways of using the kit in accordance with the present invented�eat.

All references cited in the present description, thus are included through, as against their total content, and they specifically mentioned parts.

Drawings

Fig.1: Scheme of binding at least one (or more) antibodies detection.

Fig.2: Scheme of the specific binding of the second antibody capture with partially protestirovanny or reprezentirovanii a precursor polypeptide neurotoxin and subsequent binding of at least one (or more) antibodies detection.

Fig.3: Scheme for determining binding activity of a polypeptide neurotoxin.

Fig.4: Scheme of determining the proteolytic activity of the polypeptide neurotoxin.

1. Method of determining the amount of processioning of neurotoxin polypeptide in a solution containing processionary polypeptide neurotoxin and partially processionary and/or reprezentirovanii polypeptide neurotoxin containing stages:
(a) contacting the first portion of the specified solution with a first capture antibody that specifically binds to light chains protestirovannyx, partially protestirovannyx and reprezentirovanii polypeptide neurotoxins, under conditions allowing binding of the indicated antibodies with the specified neurotoxin, with the formation, thus, the first antibody-containing�th complex,
(b) contacting the first antibody-containing complex with the antibody detection, which specifically binds to heavy chains of these protestirovannyx, reprezentirovanii and partially protestirovannyx polypeptide neurotoxins in the antibody-containing complex formed at the stage (a), whereby is formed a first set of detection
(c) contacting the second portion of the specified solution with a second capture antibody that specifically binds to the linker partially specified processioning or reprocessing of neurotoxin polypeptide, under conditions allowing binding of the indicated antibodies with partially specified protestirovanny or reprezentirovanii a polypeptide neurotoxin, with the formation, thus, the second antibody-containing complex,
(d) contacting the second antibody-containing complex with the antibody detection, whereby is formed a second set of detection
(e) determining the amount of the first and second complexes detection, formed at the stage b) and stage (d), and
f) calculating the amount of Mature neurotoxin polypeptide based on the amounts of the first and second sets of detection determined at the stage e).

2. A method according to claim 1, wherein said first antibody is immobilized capture.

3. �appliance according to claim 1, wherein said second antibody is immobilized capture.

4. A method according to claim 1, wherein the computation at the stage f) comprises subtracting a certain amount of the second complex detection of a certain number of the first complex detection.

5. A method according to claim 1, wherein said second capture antibody specifically binds to a peptide epitope having the amino acid sequence as shown in any of the sequences SEQ ID NO: 1-16.

6. A method according to claim 1, wherein said neurotoxin polypeptide is selected from the group consisting of:
(a) a polypeptide neurotoxin, as shown in any of SEQ ID NO: 17-24; and
(b) the neurotoxin polypeptide having an amino acid sequence which is at least 40% identical to the amino acid sequence of neurotoxin polypeptide according to a).

7. A method according to claim 1, where said method further comprises determining the binding activity of a polypeptide neurotoxin.

8. A method according to claim 7, containing stages:
(a) contacting portions of the solution containing the polypeptide neurotoxin, with labelled peptide, whereby is formed a complex, and
(b) definition of the specified complex, formed at the stage (a), based on the tag, where the presence or absence of the complex, or the number indicate actively linking�ti polypeptide neurotoxin in the specified solution.

9. A method according to claim 1, where said method further comprises the determination of proteolytic activity of the polypeptide neurotoxin.

10. A method according to claim 9, containing stages:
(a) contacting portions of the solution containing the neurotoxin polypeptide, with a compound having the General formula: X-pair-nitroanilide, where X is arginine or a peptide having the sequence arginine-Y, where Y represents one or more amino acids, and
(b) determining the proteolytic activity of the neurotoxin polypeptide in the specified solution on the basis of the number of released para-nitroaniline at the stage b), which correlates with the amount of neurotoxin polypeptide.

11. Method of determining the amount of processioning (active) neurotoxin polypeptide in a solution containing processionary polypeptide neurotoxin and partially processionary and/or reprezentirovanii polypeptide neurotoxin containing stages:
(a) contacting the first portion of the specified solution with a first capture antibody that specifically binds to heavy chains of the Mature polypeptide neurotoxin, partially processioning and reprocessing of neurotoxin polypeptide, under conditions which ensure the binding of the indicated antibodies with the specified Mature neurotoxin, partially protestirovanny and reprocess�included in the polypeptide neurotoxin, with the formation, thus, the first antibody-containing complex,
(b) contacting the first antibody-containing complex with the antibody detection, which specifically binds to the light chain of the specified Mature neurotoxin, partially processioning and reprocessing of neurotoxin polypeptide in an antibody-containing complex formed at the stage (a), whereby is formed a first set of detection
(c) contacting the second portion of the specified solution with a second capture antibody that specifically binds to the linkers partially specified processioning and reprocessing of neurotoxin polypeptide, under conditions which ensure the binding of the indicated antibodies with partially specified protestirovanny and reprezentirovanii a polypeptide neurotoxin, with the formation, thus, the second antibody-containing complex,
(d) contacting the second antibody-containing complex with the antibody detection, whereby is formed a second set of detection
(e) determining the amount of the first and second complexes detection, formed at the stage b) and stage (d), and
f) calculating the amount of Mature neurotoxin polypeptide based on the amounts of the first and second sets of detection determined at the stage e).

12. With�persons according to claim 11, wherein said first capture antibody immobilized.

13. A method according to claim 11, wherein said second antibody is immobilized capture.

14. A method according to claim 11, in which the calculation in stage f) comprises subtracting a certain amount of the second complex detection of a certain number of the first complex detection.

15. A method according to claim 11, wherein said second capture antibody specifically binds to a peptide epitope having the amino acid sequence as shown in any of the sequences SEQ ID NO: 1-16.

16. A method according to claim 11, wherein said neurotoxin polypeptide is selected from the group consisting of:
(a) a polypeptide neurotoxin, as shown in any of SEQ ID NO: 17-24; and
(b) the neurotoxin polypeptide having an amino acid sequence which is at least 40% identical to the amino acid sequence of neurotoxin polypeptide according to a).

17. A method according to claim 11, where said method further comprises determining the binding activity of a polypeptide neurotoxin.

18. A method according to claim 17, containing stages:
(a) contacting portions of the solution containing the polypeptide neurotoxin, with labelled peptide, whereby is formed a complex, and
(b) definition of the specified complex, formed at the stage (a), based on the tag, where the presence�e or absence of the complex, or the number indicate the binding activity of a polypeptide neurotoxin in the specified solution.

19. A method according to claim 11, where said method further comprises the determination of proteolytic activity of the polypeptide neurotoxin.

20. A method according to claim 19, containing stages:
(a) contacting portions of the solution containing the neurotoxin polypeptide, with a compound having the General formula: X-pair-nitroanilide, where X is arginine or a peptide having the sequence arginine-Y, where Y represents one or more amino acids, and
(b) determining the proteolytic activity of the neurotoxin polypeptide in the specified solution on the basis of the number of released para-nitroaniline at the stage b), which correlates with the amount of neurotoxin polypeptide.

21. A device for determining the number processioning of neurotoxin polypeptide in a solution containing:
a) system of the first capture antibody, the second antibody capture and antibody detection, where the system allows to carry out stage (a)-(e) of the methods according to any of claims.1-10; and
(b) means for calculating the amount of Mature neurotoxin polypeptide based on the amounts of the first and second sets of discovery defined by the system according to (a).

22. A device for determining the number processioning �of polypeptide neurotoxin in solution, contains:
a) system of the first capture antibody, the second antibody capture and antibody detection, where the system allows to carry out stage (a)-(e) of the methods according to any of claims.11-20; and
(b) means for calculating the amount of Mature neurotoxin polypeptide based on the amounts of the first and second sets of discovery defined by the system according to (a).

23. Set, suitable for implementing the method according to any one of claims.1-10, where the specified set contains:
a) system of the first capture antibody, the second antibody capture and antibody detection, where the system allows the implementation of stages a) to e) of the methods according to any of claims.1-10;
(b) means for calculating the amount of Mature neurotoxin polypeptide based on the amounts of the first and second sets of detection, defined using the system in accordance with a); and
c) instructions for the implementation of said method.

24. Set, suitable for implementing the method according to any one of claims.11-20, where the specified set contains:
a) system of the first capture antibody, the second antibody capture and antibody detection, where the system allows the implementation of stages a) to e) of the methods according to any of claims.11-20;
(b) means for calculating the amount of Mature neurotoxin polypeptide based on the amounts of the first and second sets of detection, defined�granted by the system in accordance with a); and
c) instructions for the implementation of said method.



 

Same patents:

FIELD: medicine.

SUBSTANCE: differential pre-operative diagnosis of the fast growth patterns of leiomyoma is ensured by measuring the relative content of CD3+CD56+CD158i lymphocytes in the peripheral venous blood of a woman with the fast-growing leiomyoma. If the derived value is equal to 0.8% or less, the true growth of leiomyoma is diagnosed, while the value of more than 0.8% shows the false growth of myoma.

EFFECT: using the given technique enables the preoperative diagnosis of the fast growth patterns of leiomyoma in the females of reproductive age that enables developing the optimum management of the patient in good time and selecting the conservative and surgical methods of treating.

1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to immunological methods of analysis and can be used for mass screening of congenital diseases in newborn children. That is ensured by immobilising in a microplate well first immune-specific components to thyrotropin, immunoreactive trypsin, thyroxin and 17α-OH-progesterone in the form of discrete microregions, placing in the well a paper disk of a dry blood sample, extracting the detectable markers from dry blood stains by adding into the well a buffer containing danazol and 8-anilinonaphthalene-1-sulfonic acid and 17α-OH-progesteron antibodies, which are bound to extracted 17α-OH-progesterone to form an immune complex with anti-species antibodies in the respective microregion simultaneously, then adding into the microplate well a reaction solution of second immune-specific components, containing a mixture of biotin-conjugated antibodies to thyrotropin, immunoreactive trypsin and thyroxin, and a 17α-OH-progesteron-protein-biotin conjugate for preparing the immune biotin-labelled complexes in the discrete microregions, removing the paper disk of the dry blood sample, adding a streptavidin and Pt-coproporphyrin conjugate to prepare the phosphorescent biotin-streptavidin complexes in the microregions on the bottom of the microplate well and detecting the label phosphoresce emission, by scanning the discrete microregions by a focused laser beam sequentially.

EFFECT: using the given method enables detecting biomarkers specifically and qualitatively in the newborn's capillary blood stain sample that enables diagnosing three newborn's congenital conditions.

10 cl, 7 dwg, 10 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to child neurology. A method includes detecting the clinical signs of a disease, measuring erythrocytes with micronuclei in a child's peripheral blood. The novel in the method for determining a degree of severity of the disease in the children with ICP is that the clinical signs of the disease are assessed by the Rivermead Mobility scale, and then an enzyme immunoassay is used to determine the qualitative content of the tumour necrosis factor (TNF-α) in the ICP children's saliva; the measured and determined values are compared to the clinical signs, and if the measured erythrocytes with micronuclei make 0.55±0.14%, saliva TNF-α increases to 13.23±5.2 pg/ml, a moderate severity is stated, while erythrocytes with micronuclei making 1.27±0.87% and more, and saliva TNF-α being 25.41±5.42 pg/ml and more show the severe disease.

EFFECT: invention enables providing the simple, non-invasive, accessible instant method for determining a degree of severity of the disease and prescribing the timely treatment.

5 dwg, 8 tbl

FIELD: medicine.

SUBSTANCE: invention represents an instant diagnostic technique for acute intestinal infections (AIIs), involving detecting indication markers of the AII aetiology with the use of laboratory immunology tests, differing by the fact that the AII aetiology is stated in children of an early age category, preferentially in the newborn children; that is accompanied by measuring the concentration of cytokine, interleukin IL-10 in coprofiltrate and diagnosing chronic placental insufficiency (CPI); a probability (P) of the bacterial AII aetiology is calculated; the value P of more than 50% testifies to the bacterial AII aetiology, while the value P being less than 50% shows the absence of the bacterial AII aetiology, and enables considering the diagnostics second stage to be necessary, which implies measuring the concentration of cytokine, interleukin IL-4 in coprofiltrate; the time of latching the newborn child to the breast is established with considering the type of feeding; that is combined with calculating a probability (P) of the viral or viral-bacterial AII aetiology, with the value P of more than 50% testifying to the viral AII aetiology, while the value being less than 50% makes it possible to state the viral-bacterial AII aetiology.

EFFECT: more accurate diagnosing of the aetiology of acute intestinal infection and simplifying the diagnostic procedure.

2 tbl

FIELD: medicine.

SUBSTANCE: venous blood and oral fluid are sampled from a patient in any sequence. An oral fluid biomarker is presented by carcino-embryonal antigen (CEA). A blood plasma biomarker is presented by neuron-specific enolase. If the oral fluid CEA is found in an amount of 196.8-318.4 ng/ml, while an amount of plasma neuron-specific enolase is 26.3-34.2 ng/ml, sarcoma of jaw bones is diagnosed. If the oral fluid CEA is found in an amount of 1228.2-1762 ng/ml with an amount of plasma neuron-specific enolase being 38.67-57.67 ng/ml, squamous cell carcinoma of jaw bones is diagnosed. The findings are used to predict a therapeutic approach to the patient.

EFFECT: invention provides high-sensitive and accurate differential diagnostic technique of maxillary and mandibular malignant new growths on the day of visit to a doctor.

2 cl, 6 ex

FIELD: medicine.

SUBSTANCE: group of inventions relates to medicine and deals with a method of determining in an individual's sample of a protein of neutrophilic origin, gelatinase-associated lipocalin (NGAL), for determination of acute kidney injury in the individual, where the predominating amount of monomer and/or heterodimer forms of NGAL protein, in comparison with the dimer form of NGAL protein, shows that NGAL protein originates from the individual's kidneys and that the individual has acute kidney injury, whereas equal or predominating amount of the dimer form of NGAL protein, in comparison with the monomer or heterodimer form of NGAL protein shows that NGAL protein originates from the individual's neutrophils and that the said individual does not have acute kidney injury; a set for determination of relative amounts of monomer, dimer or heterodimer forms of NGAL; application of the device in the said method.

EFFECT: group of inventions ensures more accurate diagnosis and therefore contributes to better directed treatment.

17 cl, 2 ex, 10 dwg, 1 tbl

FIELD: biotechnology.

SUBSTANCE: invention is a method of determining the nonspecific resistance of pathogenic microorganisms to antibiotics and the fact of the presence of bacterial biofilms on the basis of measurement of catalytic activity of phosphodiesterases cleaving the cyclic diguanosine monophosphate, with a threshold sensitivity of 50 pg/ml, comprising: 1) isolating the target-phosphodiesterase from lysed bacterial cells; 2) binding of phosphodiesterase with biotin-conjugated antibodies specific for non-catalytic domains of phosphodiesterase; 3) affinity purification of complexes formed by target-phosphodiesterase and biotin-conjugated antibody using paramagnetic particles containing neutravidin or its analogs that bind biotin; 4) interacting of the complexes of phosphodiesterase/biotin-conjugated antibody, immobilised on paramagnetic particles with complexes containing a-di-GMP in the form of G-quadruplex systems with intercalate dye, which is accompanied by decrease in the intensity while destruction of complexes of intercalate dye with c-di-GMP; 5) measurement of decrease of fluorescence upon hydrolysis with c-di-GMP and destruction of complex of c-di-GMP with intercalate dye, followed by quantitative estimation of the phosphodiesterase activity based on calibration curves made using known amounts of the recombinant enzyme of phosphodiesterase identical to the test target; 6) identification of increased level of phosphodiesterase activity detected by the test antibiotic-resistant bacterial strains capable of biofilm formation, as compared with the level of phosphodiesterase activity that can be detected for the control strains of bacteria of the same species not having the antibiotic resistance and the ability to form biofilms.

EFFECT: method enables to determine the nonspecific resistance of pathogenic microorganisms to antibiotics and to establish the fact of the presence of bacterial biofilms.

4 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to the field of immunology, namely to enzyme-immunoassay, in particular to a method of detecting forms of vascular endothelial growth factor (VEGF) with a size more than 110 amino acids in a biological sample. The method includes the following stages: contact and incubation of the biological sample with an uptake reagent, immobilised on a solid substrate, where the uptake reagent contains a monoclonal antibody, which recognises and specifically binds with residues, in quantity more than 110, from human VEGF; separation of the biological sample from the immobilised uptake reagents; contact of the immobilised molecular complex of the reagent of the uptake-target with detected antibody, which binds with VEGF domains, responsible for binding with KDR and/or FLT1 receptor, or which binds with an epitope in VEGF1-110; measurement of the level of VEGF110+, bound with reagents of the uptake, with application of means of detection for the detected antibody. Set of immune assay reagents for detection of VEGF110+ forms in the biological sample. An antibody 5C3, obtained from hybridoma 5C3.1.1 with a depositary number PTA-7737, with the said antibody 5C3 binding VEGF110+ forms, including VEGF121+. Hybridoma 5C3.1.1, deposited in ATCC with the depositary number PTA-7737, to obtain the monoclonal antibody 5C3.

EFFECT: application of the claimed invention makes it possible to increase accuracy of detecting VEGF isoforms, which must not include isoform VEGF110 and must obligatory include isoform VEGF121.

25 cl, 3 dwg, 2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention represents an immunoassay reagent which contains an agent binding to an analyte in a diluent, and glycosaminoglycan in an amount sufficient to decrease non-specific binding in an analyte sample. In the presented immunoassay reagent, the analyte is troponin I binding to the analyte; the agent is a biotin-modified anti-troponin I antibody, and glycosaminoglycan is chondroitin sulphate. Also, the invention provides a composition containing the troponin I binding agent, and chondroitin sulphate in an amount sufficient to decrease non-specific binding in the troponin I sample. What is also provided is a method of detecting the analyte in the sample wherein non-specific binding is decreased by the use of glycosaminoglycan.

EFFECT: method improvement.

5 ex, 5 dwg

FIELD: medicine.

SUBSTANCE: in the method for assessing action of biologically active substances on the antigen-antibody interaction based on sampling whole blood, stabilising it by an anticoagulant, adding a biologically active substance to the whole blood samples of the groups O(I)-AB(IV), incubating for 3-5 minutes, introducing standard monoclonal anti-A and anti-B antibodies, and 3-5 minutes later, rating the agglutination intensity relevant to the blood group.

EFFECT: improved assessment accuracy.

3 tbl

FIELD: medicine.

SUBSTANCE: method for pre-inoculation processing of a pathological material for mycobaterial recovery involves the exposure to a disinfectant, wherein the disinfectant is presented by Septustin; the processing procedure is performed by mixing aqueous solution of Septustin in the concentration of 0.5% and a pathological material in volume ratio 1:2 for 30 minutes at room temperature and followed by double 15-minute washing in normal saline.

EFFECT: invention provides high detectability with inoculation purity.

3 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: what is declared is a unified, accelerated, high-sensitive, high-specific, low labour consuming method for enhanced laboratory diagnosis of pertussis infection involving the following stages: taking a pathological material from a posterior wall of the patient's throat, preparing the clinical sample, recovering DNA from the clinical material by sorption method, performing the amplification in the isothermal environment and the electrophoresis in agarose gel, as well as a diagnostic set.

EFFECT: using the group of inventions enables identifying the pertussis agent for 4,5-5 hours from the onset of the examination directly in the patient's material depending on the stage of the disease and in various forms of the clinical course, using the group of inventions also enables establishing the diagnosis of pertussis rapidly in practical healthcare regardless of the disease length and the form of the clinical course for prescribing the timely and adequate therapy, increasing a possibility to examine the infants in pertussis-like diseases and adults with the unapparent clinical pattern, detecting the patients with pertussis in examining the centres of the pertussis infection in patient's environment as soon as possible.

2 cl, 2 ex

FIELD: medicine.

SUBSTANCE: recent comatose condition is detected; magnetic resonance imaging shows centres of cerebral structural changes; electroencephalography shows epileptiform activity, diffuse sharp waves, spikes, reduced complexes, high-amplitude low activity paroxysmal events, frequent paroxysmal events of 'peak-slow wave' and 'spike-slow wave' complexes. The EEG also shows irritant activity, local transient high-frequency beta-activity, advanced transient low-amplitude and advance continuous long-term high-amplitude activity. The presence of an aetiological agent of the disease caused by tick-borne encephalitis or the presence of encephalitis of other and uncertain aetiology is stated. The derived data are scored depending on the presence, absence and manifestations thereof. The derived data are used to calculate linear classification functions and to detect the favourable (LCF1) outcome of encephalitis without symptomatic epilepsy (SE) progression and the unfavourable (LCF2) outcome of encephalitis with SE progression. If LCF1>LCF2, the encephalitis outcome without SE progression is predicted, while LCF2>LCF1 shows the unfavourable outcome with SE progression.

EFFECT: method provides more reliable assessment of the SE progression in encephalitis that is ensured by taking into account additional EEG data and calculating the linear classification functions.

2 ex

FIELD: medicine.

SUBSTANCE: group of inventions relates to field of medicine and can be used for determination of presence of clinically relevant quantity of bacteria in donor blood or tissue. method of detecting bacterial contamination of donor blood, or blood product, or donor tissue from mammal-donor, preserved in liquid, consists in contact of sample to be analysed with a series of generic antibodies, which specifically bind with Gram-negative bacterial antigen and/or Gram-positive bacterial antigen, and detection of binding of the series with the sample. Presence of bacteria in quantity higher than 1×103 CFU of bacteria per ml of sample indicates unsuitability of donor blood, blood product or tissue for transfer to patient, with absence of said quantity of bacterial indicating suitability of donor blood, product or donor tissue for transfer to patient. Group of inventions also relates to version of said method.

EFFECT: group of inventions provides increase of accuracy and acceleration of detection of presence of clinically relevant quantity of infecting bacteria in donor blood, blood products, or in donor tissue.

10 cl, 6 dwg, 9 ex

FIELD: medicine.

SUBSTANCE: method for elution is implemented as follows: 10% tularemic, plague or brucellosis magnetic immune sorbent (MIS) 0.1ml is incubated with microbial suspensions of tularemic, plague or brucellosis agents for 30 min; the supernatant is removed; the MIS is incubated with an eluting solution 0.5 ml for 10 min; the eluate pH is reduced to the physiological value; an indirect hemagglutination reaction or a latex agglutination reaction is carried out.

EFFECT: method enables eluting the pathogen from a magnetic immobilised matrix qualitatively to be analysed by serological responses and keeping the antibody functionally active on the magnetic matrix for re-use.

3 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: method provides using a solid phase of an influenza virus vaccine for sensitisation, whereas antigen-antibody complexes formed by incubating the sensitised surface of an immunosorbent with a test serum containing the influenza virus antibodies are visualised by means of an optical contrast carbon-protein G conjugate for 10-15 minutes.

EFFECT: producing simple, high-sensitive, operative, clear and reliable diagnostic system for estimating a level of post-vaccine immunity to the influenza virus.

1 ex, 1 dwg

FIELD: medicine.

SUBSTANCE: method includes taking into account the following clinical, anamnestic and laboratory parameters (yes/no: presence of menorrhagias, presence of intrauterine surgery in anamnesis, carrying out myomectomy in hysteroscopy, level of alaninaminotransferase and asparagine aminotransferase in blood serum, content of lactobacilli in vaginal microbiocenosis, presence of enterobacteria in vaginal microbiocenosis, content of bifidobacteria in intestine microbiocenosis, presence of Staphylococcus aureus in intestine microbiocenosis, presence of fungi of species Candida in intestine microbiocenosis. data are processed. Each value is given a digital code, and they are substituted into a formula to calculate a risk of development of purulent-inflammatory complications in women with intrauterine pathology after hysteroscopy "Y", which represents the sum of values of codes of the analysed parameters. If the value Y>5, development of infectious-inflammatory complications is predicted. The method makes it possible to predict development of infectious-inflammatory diseases with accuracy up to 89%.

EFFECT: invention makes it possible to increase efficiency of predicting a risk of development of complications.

2 tbl, 2 ex

FIELD: biotechnology.

SUBSTANCE: characterised strain of influenza virus of subtype A/pochard/Siberia/249/08-MA H10N7 is isolated from cloacal elution of pochard and adapted to mice line BALB/c. The strain is deposited in the Collection of viral infections and rickettsioses of Federal budget institution of science "State Research Centre of Virology and Biotechnology "Vector" (under the registration number V-592). The strain has the high antigenic affinity for modern variants of influenza virus of subtype H10 and suitable for production of polyclonal serum for determining belonging of influenza virus to subtype H10, and can also be used as a control reference sample in assessment of specificity of the test systems based on PCR, it has lethality for mice.

EFFECT: possibility to use the strain for the study of antiviral preparations in vivo.

5 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a subtype B of a human immunodeficiency viral strain, and can be used in virology, medicine and biotechnology. The presented type 1 IV735 human immunodeficiency viral strain is deposited in the State Collection of Viruses of Federal State Institution Ivanovsky Research Institution of Virology of the Russian Ministry of Healthcare and Social Development, No. 1189. The strain possesses a stable reproductive activity. An infectious titre makes 6 lg 50% tissue cytopathic dose.

EFFECT: strain is a handy natural model for studying an antiviral activity of chemopreparations of the new generation, as well as for creating a vaccine.

1 dwg, 3 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutics. What is created is an immunologically relevant composition inducing an immune response on at least two strains and/or serotypes Streptococcus uberis, containing at least one recovered and/or recombinant protein having a specific amino acid sequence prepared by the method involving transformation of prokaryotic and eukaryotic cells or a microorganism, such as a bacterium, yeast, or fungi, by a nucleic acid construct coding the above protein, a recombinant nucleic acid molecule, a recombinant carrier and/or their immunologically relevant portion, derivative and/or analogue. A method for preparing the immunologically relevant composition. A method for identifying a protein of the bacterium Streptococcus uberis, that induces the immune response to at least two strains and/or serotypes Streptococcus uberis, involving the stages: a) identifying at least a portion of the secreted protein, superficially associated protein and/or protein min. 80% identical to a sequence of the bacterial virulence factor, b) selecting at least one protein identified at the stage a), which persists in at least two strains and/or serotypes Streptococcus uberis, c) determining an ability of at least one protein selected at the stage b) or its immunologically relevant portion, derivative or analogue to bind specifically the antibody and/or immune cells of an animal infected with the first strain and/or serotype Streptococcus uberis, and the antibody and/or immune cells of an animal infected with the second strain and/or serotype Streptococcus uberis. A method for inducing the immune response on at least two strains and/or serotypes Streptococcus uberis. And also a bovine mastitis diagnostic kit containing at least one protein having the specific amino acid sequence, the recombinant nucleic acid molecule, the recombinant carrier and the agents for detecting the antibodies.

EFFECT: using the invention enables inducing the immune response on at least two strains and/or serotypes Streptococcus uberis.

21 cl, 4 dwg, 7 tbl, 12 ex

FIELD: medicine, medicinal microbiology.

SUBSTANCE: invention relates to a method for preparing species-specific antigen C. trachomatis used for enzyme immunoassay. Chlamydia bodies are treated with amino acid chelate before treatment with detergent N-lauryl sarcosine and treatment with monosaccharide simple ether is carried out before treatment with detergent dodecyl sulfate sodium. The advantage of invention involves enhancement of species-specific activity of antigen. Invention can be used for serological diagnosis of urogenital chlamydiosis.

EFFECT: improved preparing method.

1 tbl, 3 ex

Up!