Recombinant antibodies and compositions, methods for production and uses thereof

FIELD: immunology, biotechnology.

SUBSTANCE: described are rabies virus-neutralizing antibody and fragments thereof; method for treatment of subject being affected by rabies virus using said antibody and fragments thereof. Disclosed are variants of isolated nucleic acids, encoding polypeptides bearing light and/or heavy antibody strain, respectively. Disclosed is expression vector bearing at least one from abovementioned nucleic acids. The invention makes it possible to increase subject survival after rabies virus attack.

EFFECT: method for prophylaxis and treatment of rabies virus infections.

14 cl, 1 tbl, 5 ex

 

CROSS-REFERENCE TO RELATED APPLICATIONS

This application takes priority under Provisional Application U.S. No. 60/314023, filed August 21, 20, which is incorporated herein by reference.

The technical FIELD TO WHICH the PRESENT INVENTION

The present invention relates to recombinant antibodies, including nucleic acid sequence and amino acid sequence of monoclonal human antibodies that neutralize the rabies virus.

DESCRIPTION of the prior art

Rabies is an acute neurological disease caused by infection of the Central nervous system rabies virus, a member of the genus Lyssavirus of the family Rhabdovirldae. Because of the large historical significance associated with its antiquity and daunting nature of this disease, rabies virus continues to pose a threat of infection to humans and in veterinary medicine because of the extensive distribution among the various species of wild animals. Almost all over the world for certain types of land animals endemic variants of the rabies virus, which have little in common, especially among. Despite the fact that in some regions, including the UK, Australia, Japan and many Islands, the terrestrial animal rabies is absent, virusburst and viruses, associated with rabies associated with bats, recently identified in the UK and Australia.

The rabies virus is a typical shell particle bullet-shaped form, on average, 75 to 180 nanometers. The virion consists of a single-stranded brataslava RNA genome and five structural proteins: nucleoprotein (N) molecules, phosphoprotein (NS), polymerase (L), matrix protein (M) and the viral glycoprotein (G).

Proteins N and G are antigenic determinants that allow serotypically to characterize different strains of rabies virus. Determinants of N vysokokonservativnykh different viral isolates and are therefore very easy targets for immunohistological determination infection of rabies virus using specific antibodies. On the other hand, antigenic determinants, protein G, vary significantly between strains of rabies virus. Neutralizing virus antibodies induced by vaccination inaktivirovannye virus, directed against G. Although it is clear that T-cell responses to G, N, and NS, participate in the immune response to this virus in experimental conditions, assessment of immunity to the rabies virus is usually limited to serological studies, especially as regards antibodies to neutralize the virus.

In those areas the parts around the world which is still common human disease rabies, the dog is the main reservoir of this virus that infects humans. Because rabies dogs were mostly eradicated by vaccination, foxes, coyotes, skunks, raccoons, bats, and other mammals gave shelter variants of this virus. In many areas of the reservoirs of the virus, due to wild animals continue to expand. In addition, the rabies virus can be passed from species-tanks to people or other end-hosts animals, not normally associated with rabies, such as cats, rabbits, etc.

Almost inevitably fatal when clinical symptoms appear, rabies can be prevented by immediate treatment of an infected individual by a combination of passive and active immunization. Passive immunization consists of the introduction of ready-made antibodies, neutralizing rabies virus obtained from pooled serum of individuals with immunity to rabies (human immunoglobulin against rabies; HRIG), or hyperimmunizing horses (immunoglobulin horses against rabies; ERIG). Both types of reagents pose a certain risk to recipients, including because of variable antigenic specificity and, therefore, different actions on asnie isolates of rabies virus.

HRIG is obtained from the combined human serum, therefore there is the probability that drugs HRIG can be contaminated by known or unknown human pathogens. On the other hand, being a drug foreign antigen, ERIG is associated with severe anaphylactic reactions. Mouse monoclonal antibodies specific for rabies virus, are supposed to be used for postexplosion prevention, but, like ERIG, they antigenic alien to people. This can lead to their rapid elimination from the body, and cause a possible anaphylactic reaction.

The use of human monoclonal antibodies is limited because the hybridoma cell line of human difficult to obtain, as a rule, unstable, and do not produce monoclonal antibodies of the appropriate specificity in sufficient quantities, and also quite expensive. The cost of monoclonal antibodies makes the search for more economical alternatives to obtaining monoclonal antibodies from hybridomas.

It is clearly established that the field Fab and Fab2, which contain Zahrebelny and the hinged sections of the heavy and light chains, do not protect against infection by the rabies virus. The effectiveness of the antibody in vivo depends on the full sequence, i.e. rabies active only identifying the specific antibodies. For immunoreactivity responsible constant region of the antibody. Therefore, it is a property of the constant region(s), which is necessary for protection against rabies virus. Variable regions, splanirowannya with a constant region of other antibodies, i.e. antibodies against rabies virus obtained artificially, ineffective.

There is a need for recombinant antibodies, applicable for the diagnosis, prevention and treatment of rabies infection, containing pharmaceutical compositions and providing for their use of the methods. There is a need for compositions and methods of production of such recombinant antibodies.

There is a need for recombinant antibodies, applicable for the diagnosis, prevention and treatment of infection by pathogens affecting nerve tissue containing pharmaceutical compositions and providing for their use of the methods. There is a need for compositions and methods preparation of recombinant antibodies.

To create better reagent were created human monoclonal antibodies by merging transformed by Epstein-Barr (EBV) - specific for rabies virus In human cells and mouse-human heterogenity donors. The cDNA clones encoding the heavy and light chain of the antibodies in these cells, Cohn who was TrueMobile so, to these antibodies expressibility in expressing heterologous systems. These designs make it possible to obtain controlled systems of neutralizing rabies virus human antibodies of a particular specificity, not containing potential contaminants. The present invention relates to data monoclonal antibody human, neutralizing rabies virus, sequences of nucleic acids of their heavy and light chains and amino acid sequences of data of the encoded proteins. The present invention also relates to methods of using these monoclonal antibodies as therapeutically effective postexperience prophylactic treatment of individuals exposed to rabies virus.

A BRIEF STATEMENT of the substance of the INVENTION

The present invention relates to recombinant antibodies, compositions and methods of producing such antibodies. In accordance with some aspects of the present invention, the present invention relates to recombinant rabies antibodies, and compositions and methods of producing such antibodies. In accordance with some aspects of the present invention, the present invention relates to a recombinant antibody with a specific constant region, which makes them persons of the NGOs have been effective in combating pathogens, which affect the nervous system.

In addition, the present invention relates to selected DNA sequences, recombinant vectors containing such sequences, cell host containing such vectors, and methods of producing recombinant antibodies using such host cells.

The present invention additionally relates to the use of recombinant antibodies for the diagnosis, prevention and treatment of pathogenic infections of the nervous tissue, especially rabies.

The present invention relates to molecules selected nucleic acid having the nucleic acid sequence of the heavy chain and light chain coding for the amino acid sequence of the heavy and light chain. Amino acid sequence of the heavy chain and light chain are the amino acid sequences of neutralizing rabies virus monoclonal antibody, which specifically binds with the protein of rabies virus.

The present invention relates to molecules selected nucleic acids that encode neutralizing rabies virus monoclonal antibody, which are derived from cDNA sequences of the heavy chain SEQ ID NO:1 and light chain SEQ ID NO:2.

The present invention relates to selected neutralizing virusburst monoclonal antibody man, encoded by the cDNA clones encoding the heavy and light chain antibody expressed in expressing heterologous systems and does not contain harmful impurities. In one of the embodiments of the present invention amino acid sequence selected monoclonal human antibodies that neutralize the rabies virus, represents, respectively, SEQ ID NO:3 and SEQ ID NO:4.

The present invention relates to fused gene, codereuse chimeric light chain immunoglobulin. This chimeric light chain contains a first DNA sequence encoding a variable region light chain immunoglobulin monoclonal antibodies that neutralize the rabies virus obtained using heterohybridomas cell line; and a second DNA sequence encoding a constant region of light chain of a human. The present invention relates to expressing vector, which expresses this fused gene. The next object is a host cell for expressing the vector.

The present invention relates to fused gene, codereuse chimeric heavy chain immunoglobulin. This chimeric heavy chain contains a first DNA sequence encoding a variable region of the heavy chain immunoglobulin monoclonal antibodies that neutralize the virus besent is, obtained using heterohybridomas cell line; and a second DNA sequence encoding a constant region of the heavy chain of a human. The present invention relates to expressing vector, which expresses this fused gene. An additional goal is to create a host cell for expressing created vector.

The present invention relates to the selected monoclonal antibody, neutralizing rabies virus, derived fused gene encoding a chimeric light chain of the immunoglobulin, and a fused gene encoding a chimeric heavy chain of immunoglobulin.

The present invention relates to a method of treating an individual exposed to rabies virus, by introducing the individual a therapeutically effective amount of a monoclonal human antibodies that neutralize the rabies virus, which is encoded by the cDNA clones encoding the heavy and light chains of this antibody expressed in expressing heterologous systems and does not contain harmful impurities, thereby preventing the penetration of rabies virus in the Central nervous system.

DETAILED description of the INVENTION

The present invention relates to monoclonal antibodies that specifically bind to the glycoprotein of rabies virus different is tommow. Postexperience treatment with monoclonal antibodies or a mixture of different monoclonal antibodies will neutralize the rabies virus at the level of the gate of the infection and prevent the spread of this virus in the Central nervous system (CNS). Therefore, when percutaneous or mucosal contact with the rabies virus at the site of the bite dig in and injected systemically specific to rabies virus monoclonal antibodies. Because viral replication is limited almost exclusively nerve cells, neutralization, and clearance of the virus by using monoclonal antibodies of the present invention prior to its penetration into the CNS is an effective postexplosion prevention.

The first aspect of the present invention is to create sequences of monoclonal antibodies against rabies virus. Although most variable region of Mab 57 is well known (Cheung et al., J. Virol. 66:6714-6720, 1992, which is incorporated herein by reference), constant region remains unknown. Was cloned and sequenced the complete monoclonal antibody, and the constant and variable regions. The present invention relates to new nucleotide sequence of the constant region of Mab 57, nucleotides 476-1431, which includes constant domain 1 (CH1) and the hinge region of This sequence can be used in recombinant antibodies including rabies antibodies, or recombinant antibodies, directed against other pathogens that affect the nervous tissue, such as encephalitis and herpes.

The present invention relates to a recombinant antibody, clone the genes that encode them, to vectors which include the cloned genes to cells-owners, which include data vectors. The present invention also developed methods of obtaining and using data of recombinant antibodies.

The present invention relates to recombinant antibodies derived from Mab 57. Derived from hybrid Mab 57 represent IgG2 antibody; recombinant antibodies derived from Mab 57, represent IgGI antibody. The present invention relates to recombinant antibodies derived from Mab 57, to clone the genes that encode them, to vectors which include the cloned genes to cells-owners, which include data vectors. The present invention relates also to methods of creating and using data of recombinant antibodies.

The present invention relates also to the complete sequence of the heavy and light chains rabies monoclonal antibodies Mab JA. The present invention relates to recombinant antibodies derived from Mab JA, to clone the genes that encode them, to the vectors that vklyuchayuschimisya genes and to the cells of the host, which include data vectors. The present invention relates also to methods for creation and use of recombinant antibodies.

The present invention relates also to the complete sequence of the heavy and light chains rabies monoclonal antibodies Mab JB.1. The present invention relates to recombinant antibodies derived from Mab JB.1, to clone the genes that encode them, to vectors which include the cloned genes to cells-owners, which include data sectory. The present invention also developed methods for creation and use of recombinant antibodies.

In accordance with some of the options for implementation of the present invention the recombinant antibody of the present invention is a single-chain antibody in which the heavy chain variable domain and a light chain variable domain connected by spacer elements group, preferably a peptide. Most preferable is a single-chain antibody in which the variable domain of the heavy chain is localized at the N end of this recombinant antibodies. This single-chain recombinant antibody may optionally include the effector molecule and/or signal sequence that facilitates the processing of this antibody in the cell host, in which the th receive.

Recombinant antibodies of the present invention can be used for identification of the rabies virus, for example, using immunofluorescent staining of infected cells, using Western blot turns directly or by thus and transfer of protein from the immune complexes or other immunoassay, such as binding, cross-inhibition or competitive radio - or enzyme immunoassay.

In addition, in the present invention discusses a method for the production of recombinant antibodies of the present invention. recombinant antibodies of the present invention can be obtained by using methods of recombinant DNA, involving the cultivation of the transformed host under conditions that ensure its expression and allocation of the specified antibodies.

More specifically, the present invention relates also to a method for producing recombinant antibodies, providing for the cultivation of the host, transformed with a hybrid vector containing expressing cassette comprising a promoter and DNA encoding the indicated recombinant antibody, and DNA is controlled by the specified promoter, and the selection of the indicated recombinant antibodies.

Production in vitro allows to obtain relatively pure preparations of antibodies and the scale of airavat process to produce large quantities of a desired antibody. Methods of culturing bacterial cells, yeast or mammalian cells are well known in the art and include homogeneous suspension culture, e.g. in an airlift-type reactor or in the reactor of continuous mechanical agitation or immobilized cell culture or a cell culture with the inclusion of, for example, hollow fibers, microcapsules, the agarose beads or ceramic cartridges.

Degenerate sequences are degenerate in the sense of the genetic code, according to which an unlimited number of nucleotides are replaced by other nucleotides without changing the original encoded amino acid sequence. Such degenerate sequences can be used due to the difference in their restriction sites and/or frequency of individual codons that are preferred in a particular host, in particular E. coli, for optimal expression of this recombinant antibodies.

In addition, in the present invention is considered recombinant DNA, which is a hybrid vector comprising an insert encoding a recombinant antibody described above, and, optionally, the replication origin or offline can replicate the sequence, one or more of ominantly marker sequences, controlling the expression of a sequence, the signal sequence and additional restriction sites.

Vectors typically serve two functions in cooperation with a compatible cell hosts. The first function is to ensure the cloning of nucleic acid, which encodes immunoglobulin domains, i.e. in obtaining quantities of a given nucleic acid (cloning vectors). Another function is to ensure the replication and expression of the recombinant gene constructs into appropriate host, either by saving as an extrachromosomal element or by integration into the chromosome of the host (expressing vectors). Cloning vector includes the above-described recombinant gene construct, the replication origin or offline can replicate the sequence, dominant marker sequence and, optionally, a signal sequence and additional restriction sites. Expressing the vector further includes controlling the expression of sequences required for transcription and translation of the recombinant genes.

The replication origin or offline can replicate the sequence is provided either by construction of the vector, which includes the exogenous starting point, so the y as derived from human immunodeficiency virus 40 monkeys (SV 40), or other viral source, or by using chromosomal mechanisms of the host cell.

These markers provide the possibility of selecting host cells that contain the vector. The selection markers include genes that give resistance to heavy metals, such as copper or antibiotics, such as geneticin (G-418) or hygromycin, or genes that will complementary genetic damage to the host cell, such as the lack of timedancing, hypoxanthineguanine, digidrofolatreduktazy and the like.

The signal sequence may represent, for example, the sequence precursor or secretory leader sequences that control the secretion of this recombinant antibodies, splicing signals, and the like. Examples of signal sequences that control the secretion of this recombinant antibodies are sequences derived from the gene ompA, pelB gene (spectacles) or phoA gene.

As controlling the expression of the sequences of vector DNA comprises a promoter sequence required for the initiation and termination of transcription and for stabilizing the mRNA and, optionally, enhancers and additional regulatory sequences.

You can use a wide range of p is motirola sequences depending on the nature of the host cell. Promoters that are strong and, at the same time, well-regulated, are the most suitable. Sequence for initiation of translation are, for example, the sequence of the Shine-Dalgarno. Sequences necessary for the initiation and termination of transcription and for stabilizing the mRNA, usually are, respectively, in the non-coding 5'-regions and 3'-regions of viral or eukaryotic cDNA, for example, from the expressing of the owner. The enhancers and stimulating the transcription of DNA sequences of viral origin, e.g. derived from the human immunodeficiency virus monkeys, Polyanovo virus, human papilloma virus of cattle or sarcoma virus, moloni, or genomic origin, especially the mouse.

Different segments of the DNA vector DNA are connected by stitching, i.e. they are continuous and are in a functional relationship with each other. Examples of vectors which are used for replication and expression in E.coli strain are bacteriophages, for example derivatives of lambda bacteriophages, or plasmids, such as, for example, in particular, as a plasmid ColE1 and its derivatives, for example, RMV, pSF2124, pBR317 or pBR322 and lamidi derived from pBR322, such as pUC9, pUCKO, pHRi148 and Lc24. Suitable vectors contain a complete replicon, mA is Cerny gene sequence, recognizing restriction endonuclease so that the foreign DNA and, if necessary, expressing the control sequence can be embedded in these sites, as well as optional signal sequences and enhancers.

The promoters of microorganisms are, for example, a strong left promoter PLbacteriophage X, which is controlled by a temperature-sensitive repressor. Suitable are also the promoters of E. coli, such as lac (Lac)promoter, adjustable lac-repressor and induced by isopropyl-beta-D-thiogalactoside, trp (tryptophan)-promoter, adjustable trp-repressor and induced by, for example, when tryptophan starvation and tac (a hybrid trp-lac promoter), adjustable lac-repressor.

Vectors applicable for replication and expression in yeast contain yeast replication origin and genetic marker selection for yeast. One group of such vectors include the so-called ars sequence (Autonomous replicating sequence) as the starting point of replication. These extrachromosomal vectors are saved in a yeast cell transformed and replicated autonomously. In addition, it is possible to use vectors that contain all or part of the 2 ám plasmid DNA in Saccharomyces cerevisiae. Such vectors will be integrated the change by recombination in already existing in the cell of 2 μm plasmids or replicate autonomously. 2 micron sequences are particularly suitable for achieving high frequency transformation and a large number of copies.

Controlling the expression of the sequence that are applicable for expression in yeast are, for example, the control sequence vysokoagressivnyh yeast genes. Therefore, it is possible to use promoters of genes RP1, the ADHI or ADHII, gene, acid phosphatase (RNA or RNA), soltahanova gene or promoter involved in the glycolytic pathway, such as the promoter of genes enolase, glyceraldehyde-3-povetkins (PGK), hexokinase, piruvatcarboksilazy, phosphofructokinase, glucose-6-fortismere, 3-phosphoglyceromutase, pyruvate kinase, triosephosphate, phosphoglucomutase, and glucokinase.

Vectors applicable for replication and expression in mammalian cells, preferably contain promoting sequences obtained from DNA of viral origin, such as human immunodeficiency virus 40 monkeys (SV40), rous sarcoma virus (RSV), adenovirus 2, human papilloma virus of cattle (BPV), BK-mutant of papovavirus (BKV) or cytomegalovirus (CMV), mouse or human. Alternatively, the vectors may include the promoters of the products of expression of mammals, such as actin, collagen, myosin, and the like, or the native promoter and control sequences, the cat is that usually associated with the desired gene sequence, ie promoter H-chain or L-chain immunoglobulin.

Some preferred vectors are applicable for both prokaryotic and eukaryotic hosts and are based on viral replication systems. Particularly preferred vectors include the promoters of the human immunodeficiency virus monkeys, for example pSVgpt or pSVneo, including additional enhancer, for example, the enhancer is usually associated with the immunoglobulin sequences of the gene's particular enhancer N - or L-chain Ig mouse.

Recombinant DNA encoding the recombinant antibody of the present invention, can be obtained, for example, by culturing the transformed host cell, and optional selection, DNA.

In addition, the present invention relates to a cell host transformed with the above recombinant DNA, namely the cell host transformed by a DNA that encodes a heavy chain and/or DNA that encodes a light chain recombinant antibodies, in particular, to the cells of the host, transformed with DNA that encodes a single-chain recombinant antibody.

In particular, the present invention considers a host cell, transformed with a hybrid vector containing expressing cassette comprising a promoter and DNA encoding a recombinant antibody.

In addition, the present invention relates to the cell host transformed hybrid vector containing expressing cassette comprising a promoter functionally linked to the first DNA sequence that encodes a signal peptide attached in the open reading frame to a second DNA sequence that encodes a recombinant antibody.

Examples of suitable hosts represented by microorganisms that do not contain or contain few enzymes or modifying enzymes, such as bacteria, in particular strains of Escherichia coli, for example E. coli X1776, E. coli Y1090, E. coli HB 101, E. coli W3110, E. coli HB 101/LM1055, E. coli JA 221, E. coli DH5.alpha., E.coli K12, or E.coli strain SS, Bacillus subtilis. Bacillus stearothermophilus, Pseudomonas, Haemophilus, Streptococcus and others, as well as yeasts, for example Saccharomyces cerevisiae, such as S.cerevisiae 3RF 18. In addition, appropriate cell hosts are represented by the cells of higher organisms, in particular stable continuous cell lines of human or animal, for example lung fibroblasts L132 of a human embryo, cells Bowes malignant melanoma, HeLa cells transformed by SV40 virus kidney cells of the African green monkey COS-7 or cells Chinese hamster ovary (Cho) or cells of lymphoid origin, such as simfonie, myeloma, hybridoma, trianee or quadrom the s cells, for example, PAI, S2/0 or H-Ad.

The present invention relates also to methods of producing a transformed host cells, in which a suitable recipient cell host as described above, transformed with a hybrid vector according to the present invention, and the resulting transformed cells are selected. The transformation of the microorganisms is carried out, as described in their published work, such as S.cerevisiae (A.Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978), B. subtilis (Anagnostopoulos et al., J. Bacteriol. 81:741, 1961), and for E. coli (.Mandel et al., J. Mol. Biol. 53:159, 1970).

Accordingly, the method of transformation of E.coli cells include, for example, pretreatment of cells with ions of CA2+in order to make possible the capture DNA, and incubation with this hybrid vector. You can then make the selection of transformed cells, for example, by migrating cells on the selective growth environment that allows you to separate the transformed cells from the parent cell, depending on the nature of the marker sequences of the vector DNA. It is preferable to use growth environment, which prevents growth of cells that do not contain the vector. Transformation of yeast include, for example, stage enzymatic removal of the shell yeast cells using glucosides, processing obtained is of spheroplasts data vector in the presence of polyethylene glycol ions and Ca 2+and the regeneration of the remote cell membrane by the conclusion of the processed spheroplasts in agar. Preferably, the agar for the regeneration of the get method, which makes possible the regeneration and selection of transformed cells as described above, at the same time.

Transformation of the cells of higher eukaryotes, such as mammalian cell lines, preferably carried out successfully by transfection. Transfection carried out by traditional methods such as calcium phosphate precipitation, microinjection, fusion of protoplasts, electroporation, i.e. the introduction of DNA by means of an electrical impulse, which temporarily increases the permeability of cell membranes, or in the presence of auxiliary compounds such as diethylaminoacetate, dimethylsulfoxide, glycerol or polyethylene glycol, and the like. After surgery transfection transfetsirovannyh cells identify and selectyou, for example, by culturing in a selective medium is selected depending on the nature of the selective marker, for example in the standard culture medium, such as a modified Dulbecco Wednesday Needle (DMEM), minimum essential medium, medium RPMI 1640 and the like, containing, for example, the appropriate antibiotic.

Recombinant antibodies, in accordance with the present invention, can be used on lesofat for qualitative and quantitative determination of the presence of rabies virus. In General, recombinant antibodies, in accordance with the present invention can be used in any known immunopharmacol analysis, which depends on the binding interaction between antibodies and antigens rabies. Examples of such analyses are radio-, enzyme, fluorescent, chemiluminescent, immunoprecipitations, latexotica.com, and hemagglutination immune analysis, and, in particular, how the immune staining.

In accordance with the present invention antibodies can be used as such or in the form conjugated with enzyme derivatives in enzyme-linked immunosorbent assay. You can use any known modification enzyme immunoassay, for example, liquid-phase (homogeneous) enzyme-linked immunosorbent assay, solid phase (heterogeneous) enzyme-linked immunosorbent assay, one-step enzyme-linked immunosorbent assay or two-stage (sandwich) immunoassay analysis with direct or indirect (competitive) the presence of rabies virus.

An example of such an immunoassay is a sandwich enzyme-linked immunosorbent assay, in which a suitable carrier, such as a plastic surface of a microtiter plate or test tube, for example polystyrene, polypropylene or polyvinyl chloride, glass or plastic beads, fil is revelou paper, dextranase and similar cellulose acetate or nitrocellulose sheets, magnetic particles or the like, cover the monoclonal antibody of the present invention a simple adsorption or optionally after activation of the carrier, for example with glutaraldehyde or bromine cyan. Then add the tested solutions containing the rabies virus, and, finally, the recombinant antibodies of the present invention that includes the detected enzyme, such as alkaline phosphatase. The number of rabies virus in the test solution is directly proportional to the amount of bound recombinant antibodies and is determined by adding the enzyme-substrate solution. The enzyme-substrate reaction gives, for example, a change in color that can be seen with the naked eye or with optical measuring devices.

In accordance with the present invention antibodies can be used as such or in the form of radioactively labeled derivatives in radioimmunoassay analysis (RIA). As described above for immunofermentnyh analyses, you can use any modification of the radioimmunoassay analysis.

Testing carried out in a manner analogous to the above immunoassays using a radioactive label, such as125I, instead of the enzyme label. The amount of immune complex, Bratanova with the appropriate number of rabies virus, present in the test solutions is determined by measuring the radioactivity of the immune complex.

For immunoablative slices frozen or stored at cryogenic temperatures biopsy material or enclosed in paraffin tissue sections treated with a solution containing the recombinant antibody of the present invention comprising a detectable enzyme. Associated recombinant antibody is detected by treatment with a suitable enzyme substrate, preferably an enzyme substrate, which gives a precipitate of solid particles (dye) on the site of present recombinant antibodies of the present invention. Instead of a recombinant antibody comprising an enzyme, it is possible to use recombinant antibody comprising streptavidin and a solution of Biotin-enzyme conjugate, which leads to increased enzyme concentration at the site of this antibody and, therefore, increases the sensitivity of the method is immune staining. The precipitated solids enzymatic substrate detected by viewing under a microscope, for example using a fluorescent microscope, or by scanning the optical density at a particular wavelength of this dye.

Using, in accordance with the present invention, recombinant antibodies, as described above, DL is the definition of rabies virus also includes other immune tests, known per se, for example, immunofluorescent assays, latex agglutination with latex particles coated with antibody or antigen, the hemagglutination of red blood cells coated with antibody or antigen analysis of the evanescent wave light using antibody-coated fiber or other immunosensors direct actions that transform the event binding in electrical or optical signal, or the like.

The present invention also covers test kits for the qualitative and quantitative determination of the presence of rabies virus comprising a recombinant antibody of the present invention and optional excipients, positive and/or negative controls, buffers, instructions and descriptions sample results.

In addition, the recombinant antibodies of the present invention is used for prevention of the disease rabies in patients suspected of possible contact with the rabies virus, or to treat patients who are infected with rabies.

Therefore, the present invention relates also to pharmaceutical compositions comprising a therapeutically effective amount of recombinant antibodies in accordance with the present invention, and a pharmaceutically acceptable carrier. Preferred are supplied with the ski compositions for parenteral use. Compositions for intramuscular, subcutaneous or intravenous use are, for example, isotonic aqueous solutions or suspensions, optionally obtained shortly before use of lyophilized or concentrated preparations. Suspensions contain oil as the oil component of the vegetable, synthetic or semi-synthetic oils, customary for injection purposes. Data pharmaceutical compositions may be sterilized and may contain auxiliary substances, such as preserving, stabilizing, wetting, emulsification or solubilization of these ingredients, salts for regulating the osmotic pressure, buffers and/or compounds that regulate the viscosity, for example nitrocarburization, carboxymethyl cellulose, sodium carboxymethyl cellulose, dextran, polyvinylpyrrolidine or gelatine.

The pharmaceutical compositions of the present invention contain from about 0.01% to about 50% active ingredients. They can be in the form of dosage units, such as ready for use ampoules and vials, or in the form of lyophilised solids.

In General, prophylactically and therapeutically effective dose for mammals is approximately 0.5-250 μg of recombinant antibodies of the present from which retene per kg of body weight depending on the type of antibody, status of the patient and patterns of use. The specific scheme introduction and appropriate dose should choose your doctor, taking into account the individual characteristics of the patient, status of the disease, the type of tumor, and the like. The pharmaceutical compositions of the present invention is produced by methods known in the art, such as conventional mixing, dissolving, methods of preparation of medicines or liofilizirovanny. Pharmaceutical compositions for injection process, fill ampoules and vials and sealed under aseptic conditions in accordance with methods known in the art.

In some embodiments, implementation of the present invention compositions and/or methods are related to or antibody-based test mixtures, combined with one or more antibodies. In preferred embodiments, the implementation of the present invention

These blends contain two or more antibodies of the present invention.

EXAMPLE

Example 1

Cells

Used for hybridization In human cells obtained from the peripheral blood of 5 donors between 7-21 days after the third dose of the primary rabies vaccination and 5 donors, immune to rabies, in 10-21 days after re-introduction of the vaccine. In all cases, use the t vaccine Rabivac™ vaccine diploid human cells (viral strain Pitman Moore 1503-3M, Behringwerke, Marburg, FRG). All donors were negative in tests for HIV and hepatitis C. Hybrid heteromyinae cells SHM-D33 mouse-human, used as partners for mergers in hybridoma (Teng, N.N. et al., Proc. Natl. Acad. Sci. USA 80, 7308, 1983), and leukocytes monkeys, transformed virus B95-8 Epstein-Barr (EBV), used as a source of EBV (Henderson et al., J. Exp. Med. Vol.76, R, 1977), were obtained from ATS (Rockville, MD).

Viruses rabies

To assess the ability of the antibody preparations to neutralize different strains of rabies virus, use antigenically distinct fixed laboratory strains, as well as two representative virus of street rabies. Fixed strains Evelyn-Rjkitnicki-Abelseth (ERA), the standard strain of a virus infection, or adapted to mouse brain (CVS-24)or cell culture (CVS-11) and Pitman-Moore (PM)derived from the virus collection of Thomas Jefferson University. The rabies virus silver bats (SHBRV)that are associated with the most recent cases of rabies in the United States of America, and the virus of street rabies coyotes/the rabies virus in dogs Mexico (COSRV), which is representative viruses of rabies dogs obtained as described (Morimoto et al., Proc. Natl. Acad. Sci. USA, vol.93, p.5653, 1996). Dedicated cleaning in the Rus and the preparation of the glycoprotein (G), and nucleoprotein (N), described elsewhere (Dietzschold et al., World Health Organization, Geneva, p.175, 1996).

EBV - transformation of human PBL

Menagerie cells of peripheral blood (RMS) was isolated from whole blood by centrifugation in density gradient Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ), which is described in detail elsewhere (Plebanski et al., Immunology Vol.75, R, 1992). Then T cells Deplete negative selection using magnetic beads coated with a monoclonal antibody against CD2 (Dynal Inc., Lake Success NY), and the hub of magnetized particles (Dynal). CD-2-negative cells, primary b cells, collect and immortality, as described previously (Swaminathan, 1992). Briefly, cells B95-8, cultured to confluence in RPMI1640(Gibco BRL Life Technologies, Grand Island NY) supplemented with 10% serum fetal cow (FBS; Gibco), are lysed by freeze-thawing in dry ice to release intracellular EBV. The supernatant, containing EBV, clarify by centrifugation at 1000 rpm for 10 min and filtered through 0.45 µm filter. The virus concentrated by centrifugation at 8000 rpm for 2 h at 4°C. 7×106B-cells (suspended in 1 ml of culture medium for B95-8) incubated at 37°C for 2 h with virus obtained from 25 ml of cells B95-8. After infection the cells twice washed with culture medium, seeded in 96-well Krugloe the nye die for micrometrology (Nunc, Fisher Scientific, Pittsburg, PA) at a concentration of 1×104cells/well, and cultured at 37°C in a humid atmosphere with 5% CO2and 95% air.

Detection of heterohybridomas mouse-human

After EBV transformed cell lines and their cultured for approximately 4 weeks, collect the supernatant and tested in ELISA for the presence of antibodies specific for rabies virus. Positive wells are initially transferred into 1 ml, then 2 ml of culture (48 - and 24-hole plates, Nunc), and the resulting supernatant analyze then in a quick test on the inhibition of fluorescence emission (RFFIT) (Hooper, ASM Press, WA p.775, 1997). Cell lines producing neutralizing antibody hybridized cells SHM-D33 (ADS, catalogue number CRL1668) as follows. An equal number SHM-D33 and EBV-transformed cells (approximately 5×106each) are placed together in a sterile polystyrene round-bottom tube Falcon, Fisher Scientific) and centrifuged at 1000 rpm for 10 minutes, the Cells washed twice serum-free medium and the precipitate cells resuspended in 100 µl medium.

The tubes are heated at 37°With water bath for 1 min and then added dropwise within 45 sec 0.5 ml of heated 37° (C) 50% (wt./about.) polyethylene glycol (Sigma, Chemical Co., St. Louis, MO, catalog number P-7181), heated gently shaking the tube. Then reacts the Yu merge stop, slowly Prilepa 3 ml of serum-free medium for 30 sec, followed by addition of 9 ml of this medium for 30 seconds. The tubes leave at room temperature for 8 min and then incubated for 2 min at 37°With water bath. Then these cells are centrifuged at 500 g for 3 min and the precipitate cells resuspended in 30 ml of Iscove modified environment Dulbecco (IMDM; Gibco)containing 10% FBS and 0.4 µm of aminopterin (Gibco) and 10 μm ouabain (Sigma) for selection of cells that are not hybridized. Cell suspensions were seeded in 96-well round-bottom plates to micrometrology concentration of 1×104cells per well and incubated as described for cell lines.

When the colonies heterogenity cells will be formed (approximately 6-week culture), supernatants tested in ELISA and RFFIT education specific to rabies virus antibodies. Antitelomerase cell clone, at least three times using limiting dilution in tablets for micrometrology. Cells and titrated 96-well round-bottom tablets 2-multiple dilutions, starting with 4 cells per well. Cells from the wells, on average, cells containing 0.25 or less, multiply for the collection of the supernatant and analyze further.

Analysis specific to rabies virus antibodies in ELISA

The specificity of antibodies and isotype is anywayt in solid-phase ELISA. Plates (PolySorb™, Nunc) are covered at room temperature in a humidified chamber overnight 5*g/ml ERA rabies virus, glycoprotein, or nucleoprotein diluted in phosphate-buffered saline (PBS). Then these tablets are blocking with 5% milk powder in PBS and washed them in PBS containing 0.05% tween20(PBS-Tween) before adding supernatant samples.

After incubation at room temperature for 2 h the resulting tablets is washed with PBS-Tween to remove unbound primary antibodies for 1 h at room temperature add various enzyme-conjugated or biotinylated secondary antibodies that are specific to different heavy chain isotypes of human rights. Secondary antibody detects either by formation of a soluble end product in the environment after adding the appropriate substrate (3,3',5,5'-tetramethylbenzidine (TMB) in phosphate-citrate buffer, or para-nitrophenylphosphate (PNPP) in 0.1 M glycine buffer (Sigma) or after addition of avidin-alkaline phosphatase (30 min at RT) and PNPP substrate. The reaction of the peroxidase-TMB stopped by the addition of 2 M H2SO4. The absorption values read in the spectrophotometer for the microplate (Biotek, Winooski, VT) at 450 for TMB-product and at 405 nm for PNPP reaction.

RFFIT

Supernatant samples of each of transformer the private cell lines analyzed in the presence of antibodies, neutralizing rabies virus, using changes quick test on inhibition of fluorescent glow (RFFIT)as Previously described (Hooper, ASM Press, WA p.1997). Supernatant samples (50 ál) were diluted in 96-well round-bottom plates (Nunc). The cultivation of rabies virus, which causes 80-90% infection of indicator cells is added to each test sample, and these tablets are incubated at 37°C for 1 h Negative environment and positive for rabies immune serum control samples included in each analysis. After incubation, each well add cells cub Chinese hamster (KSS) at a concentration of 30 ul of 1.8×l06cells/ml and the culture incubated overnight at 37°C. Then the tablets once washed with ice-cold PBS and fixed with ice-cold 90% acetone for 20 min at -20°C. After fixation, the acetone is removed and the tablets are dried in the air. For detection of infected KSS cells in each well add 45 min at 37°With 40 ul FITC monoclonal globulin (Centrocor, Malvern PA) against nucleoprotein rabies. Then these plates are washed three times with distilled water and viewed under a fluorescent microscope.

The highlight for the purification of antibodies using chromatography on affinity

IgG1-antibody allocate purification using column protein A (rProtein A Spharose TMFast Flow, Amersham Pharmacia Biotech). Briefly, supernatant lighten filtered through 0.45 µm membrane and adjusted pH to 8.0 with 1N NaOH. Supernatant passed through this column with a linear flow rate of approximately 100 cm/hour. After washing in PBS (pH 8) antibodies elute from the column using a solution of 0.1 M citric acid and then cialiswhat against PBS.

IgG3-secrete antibody purification using column protein G (Protein G SepharoseTMFast Flow, Amersham Pharmacia Biotech). The supernatant, containing IgG3, clarify by filtering through 0.45 µm membrane and the pH was adjusted to 7.0 using 1N NaOH. The supernatant is passed through this column with a linear flow rate of approximately 11 cm/hour. After washing with PBS, the antibody elute from the column using 0.1 M glycine buffer, pH 3.0, and then cialiswhat against PBS.

IgM-antibody are cleaned using mannan-binding protein and modification of a previously described method (Nevens et al., J. Chromatogr, vol.597, p.247, 1992). In short, containing IgM supernatant treated with EDTA, adjusted to pH 8.0 with 1M NaOH, filtered and cooled to 4°S. Mannan-binding protein-a agarose (Sigma), washed in the column with 4°With a buffer consisting of 0.1 M Na**CO3/0.5 M NaCl, pH 8,3, then add to the supernatant and incubated in the column for 15 min at 4°C. Then this column protivotumanki volumes of binding buffer and brought to a CT scan within 1 hour IgM elute from the column binding buffer at RT and cialiswhat against PBS.

Protein concentration cialisovernight drugs antibodies determined using analysis of protein detection (Bio-Rad Labcratories, Hercules, CA) as follows. 100 μl of sample is added to 5 ml of diluted 1/5 concentrate dye reagent at RT for 10 minutes. In each analysis include negative PBS control and different protein standards serum albumin cattle. After incubation, the samples are read in a spectrophotometer at 595 nm. Protein concentrations of the tested samples count towards the absorption of BSA standards. The degree of purification of all drugs antibodies assessed by electrophoresis in a 12.5% polyacrylamide gel under reducing conditions (SDS-PAGE). Dedicated cleaning antibodies demonstrate in SDS-PAGE, two major bands corresponding with the selected heavy and light immunoglobulin chains.

Production, isolation and sequencing of cDNA clones

Total RNA extracted from JA-hybridoma cells by using RNAzol B (Biotecx Laboratories, Houston). The reaction of reverse transcription is carried out at 42°C for 1 h with reverse transcriptase virus myeloblastosis (Promega) and oligo(dT)-primer. Part obratnoosmoticheskih products are subjected to amplification in a polymerase chain reaction (PCR), IP is by using the primers specific for the heavy chain of IgG-HF1-primer (5'-ACCATGGAGTTTGGGCTGAG-3' (SEQ ID NO:5), the start codon; underlined, catalogue No. Y14737), and IgG-HR2-primer (5'-ACTCATTTACCCGGGGACAG-3' (SEQ ID NO:6), the stop codon is underlined, catalogue No. Y14737) or primers specific for the light chain: IgG-LF5-primer (5'-AGCATGGAAGCCCCAGCTCA-3' (SEQ ID NO:7), the start codon; underlined, catalogue No. M), and IgG-LR2-primer (5'-CTCTAACACTCTCCCCTGTTG-3' (SEQ ID NO:8', the stop codon is underlined catalogue No. M). Amplification is carried out for 35 cycles of denaturation at 94°C for 60 seconds, annealing at 50°C for 60 seconds, and polymerization at 72°C for 90 seconds with Taq-DNA polymerase (Promega). The data of PCR products (1,4 TPN for the heavy chain, 0,7 TPN for light chain) are cleaned and is sequenced using AmpliTaq-set to cycle sequencing (Perkin-Elmer) with specific primers. The obtained PCR products clone in the TA-cloning vector, pCR2.1 (Invitrogen). The cloned cDNA of the heavy chain and light chain is sequenced using AmpliTaq-set to cycle sequencing (Perkin-Elmer) with specific primers.

Coding sequences of a monoclonal antibody neutralizing rabies virus

cDNA monoclonal antibodies, and complementary to her sequences are nucleic acids, monoclonal antibodies, generated in the present from which britanii. In a specific embodiment of the present invention the DNA sequence of the monoclonal antibody create for the heavy chain (SEQ ID NO:1) and light chain (SEQ ID NO:2) monoclonal antibody from clone JA, and therefore do not have any introns.

The present invention relates also to the single-stranded oligonucleotides for use as primers for PCR, which amplificare fragment containing the sequence of monoclonal antibodies, for example, variable or hypervariable region of this monoclonal antibody. Oligonucleotide having the sequence hybridizes plot, at least 8 nucleotides, gene monoclonal antibodies, and the other oligonucleotide back with a complementary sequence to the right on the same chain gene monoclonal antibodies so that each oligonucleotide primer synthesizes direction to another. These oligonucleotides contain preferably in the range 10-35 nucleotides in length.

The present invention relates to a full-sized cDNA sequences for the heavy and light chains of monoclonal antibodies from heterohybridomas clone JA (respectively, SEQ ID NO:1 and SEQ ID NO:2), and the encoded polypeptides of 1-474 amino acids for the heavy chain (SEQ ID NO:3) and amino acids 1-234 for the light chain (SEQ ID NO:4).

In oncletom embodiment described here the implementation of the present invention, the present invention relates to a nucleic acid sequence of the monoclonal antibody of heterohybridomas clone JA. In a preferred, but not limited aspect of the present invention this heterohybridomas clone JA is the source of cDNA monoclonal antibodies.

Functional equivalents monolocali antibodies, neutralizing rabies virus

The present invention also includes functional equivalents of antibodies presented in this description. Functional equivalents have binding characteristics comparable to the binding characteristics of the considered antibodies, and include, for example, chimeric, and single-chain antibodies, and fragments thereof. Methods for such functional equivalents are disclosed in PCT application WO 93/21319, in European Patent Application No. 239400; PCT application WO 89/09622; European Patent Application 338745; and European Patent Application EP 332424.

Functional equivalents include polypeptides with amino acid sequences are basically the same as the amino acid sequence of the variable or hypervariable regions of the antibodies of the present invention. "Basically the same" amino acid sequence shown here in the form of a sequence with at least 70%, preferably at least the Colo 80%, and most preferably at least about 90% homology to another amino acid sequence, as determined using the search method is FAST, according to Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85, 2444-2448, 1988. Chimeric antibodies have constant regions derived substantially or exclusively from constant regions of human antibodies and variable regions derived substantially or exclusively from the sequence of the variable regions of monoclonal antibodies each stable heterohybridomas (Champion, J.M., et al., Journal of Immunological Methods, 235 31-50, 2000).

Single-chain antibodies or Fv fragments are polypeptides which consist of variable regions of heavy chain antibodies, coupled with the variable region of the light chain by means of a connecting linker or without him. Thus, Fv includes a full site binding of this antibody.

In addition, functional equivalents include fragments of antibodies that have the same, or substantially the same binding characteristics to that of the whole antibody. Such fragments may contain one or more Fab fragments of the F(ab').sub.2 fragment. Preferably, if all the data fragments or antibody-based test contains areas that define the complementarity of the whole antibody, although fragments containing individual that is their area, for example, three, four or five areas that define complementarity, are also functional. Functional equivalents are members of the immunoglobulin class IgG and its subclasses, but can constitute or can combine any of the following immunoglobulin classes: IgM, IgA, IgD, or IgE, and their subclasses. Heavy chains of different subclasses, such as IgG-subclasses that are responsible for different effector functions and, therefore, choosing the desired constant region of the heavy chain, get a chimeric antibody with a desired effector function. Preferred constant regions are gamma 1 (IgGI), gamma 3 (IgG3) and gamma 4 (IgG4). Constant region light chain can be a Kappa or lambda type.

The immunoglobulins of the present invention can be monovalent, divalent or polyvalent. Polyvalent immunoglobulins represent dimers (HL)derived from chimeric heavy chain, linked through disulfide bridges with the chimeric light chain. Bivalent antibodies are tetramera (H2L2derived from the two dimers associated at least one disulfide bridge.

Standard methods for obtaining recombinant DNA

Standard methods for obtaining recombinant DNA described in Sambrook et al., "Molecular loning" (Molecular Cloning), Second Edition, Cold Spring Harbor Laboratory press (1987) and Ausubel et al. (Eds) "Current Protocols in Molecular Biology" (current Protocols in Molecular Biology, Green Publishing Associates/Wiley-Interscience, New York (1990).

In short, choose the appropriate source of cells containing nukleinovokisly molecules which Express the desired DNA, such as antibodies or equivalent DNA antibodies. Total DNA obtained using the standard procedure from the appropriate source. Total RNA used for direct synthesis of cDNA. Standard methods RNA extraction and cDNA synthesis are presented in the standard manuals of molecular biology, such as that described above.

The obtained cDNA is possible to amplify by known methods. For example, the obtained cDNA can be used as template for amplification in polymerase chain reaction (PCR); see Saiki et al., Science 239, 487, 1998 or Mullis et al., U.S. patent No.*83195. The nucleotide sequence of primers for PCR amplification derived from a known sequence that is amplified. Oligonucleotides are synthesized by methods known in the art. Suitable methods include the methods described in Caruthers in Science 230, 281-285, 1985.

In PCR amplification using the left and right oligonucleotides. To optimize conditions for each pair of primers in the CE is provided with the standard operating procedures. The obtained PCR product is analyzed, for example, by electrophoresis for cDNA with the correct size, corresponding to the sequence between the primers.

Alternatively, the coding region can amplify two or more overlapping fragments. Overlapping fragments of create to enable the restriction site that allows you to mount the intact cDNA of data fragments.

In order to distinguish protein coding full-sized region of the heavy and light chains each monoclonal antibody from each heterohybridomas cell lines, for example, the left PCR oligonucleotide primer should be complementary to the sequence 5'-end, covering the start codon ATG and at least 5-10 nucleotides to the left from the start codon. Right PCR oligonucleotide primer must be complementary sequence at the 3'-end of the desired DNA sequence. The desired cDNA encodes the whole plot heavy and light chains of each monoclonal antibody, including the stop codon.

Amplificare cDNA encoding these antibodies or antibody-based test, or equivalents, can also be replicated in a variety of cloning vectors in different host cells. Such a host cell may be prokaryotic or eukaryotic.

Vecto is, in which spiceroads cDNA monoclonal antibodies may include chromosomal sites, achromosomal and synthetic DNA sequences. Some suitable prokaryotic cloning vectors include, but are not limited to, plasmids from E. coli, such as colE1, pCR1, pBR322, RMV, pUC, pKSM, and RP4. Prokaryotic vectors include, but are not limited to, derivatives ragovoy DNA such as M13 and other single-stranded DNA of filamentous phage.

The vector containing the cDNA of a monoclonal antibody which is expressed, transferout in a suitable cell host, as described below. Cell-host support in the appropriate culture medium and exposed to conditions under which this cell and the vector is replicated.

Chimeric antibodies

In General, the chimeric antibodies are produced by obtaining for each component of the chimeric immunoglobulin light and heavy, fused gene comprising a first DNA that encodes at least a functional part, specifically neutralizing rabies virus human, preferably a glycoprotein, a variable region of a human immunoglobulin, concatenated (for example, functionally reconstructed variable region with the attached scope) with a second DNA coding for at least part of a constant region and the human immunoglobulin. Each slit mounted gene in the expression vector or embed it. Recipient cells that can Express the gene products, transferout then with the help of these genes. Transfetsirovannyh recipient cells are cultured in conditions, catorce allow to Express the built-in genes, and downregulation of immunoglobulin or immunoglobulin chain to retrieve.

The genes encoding the variable region of immunoglobulin heavy and light chains derived from lymphoid cells, which produce antibodies that neutralize the rabies virus. For example, heterohybridomas cell lines that produce monoclonal antibody against the glycoprotein of rabies virus, are a source of immunoglobulin variable regions for the chimeric antibodies of the present invention. A constant region derived from antibody productive human cells using standard cloning techniques. Alternatively, because the genes are represented by two classes of light chains, these classes of heavy chain clone human-derived constant region affected easily accessible from these clones. Fragments of binding of chimeric antibodies such as F(ab').sub.2 and Fab fragments, obtained by constructing a chimeric gene of the heavy chain in the reduced form. For example, a chimeric gene, kodiruyushchikh heavy chain F(ab').sub.2, must include a DNA sequence encoding the CH1domain and hinge region of this heavy chain. Alternatively, such fragments can be obtained by enzyme treatment of chimeric immunoglobulin. For example, treatment with papain or pepsin can form, respectively, Fab or F(ab').sub.2 fragments.

Preferably, the fused genes encoding the chimeric heavy and light chains, or portions thereof, are mounted in two different expressibly vector, which can be used to cotransfection recipient cells. Each vector contains two selective gene, one for selection in bacterial system, and the other for selection in eukaryotic system, and each vector has a different pair of genes. These vectors allow for the accumulation and amplification fused genes in a bacterial system, and the subsequent cotransfection eukaryotic cells and selection received cotransfection cells. Examples of selective genes for the bacterial systems include, but are not limited to, genes that give resistance to ampicillin and genes that give resistance to chloramphenicol. Two selective gene for selection of eukaryotic transfectants are preferred, but are not limited to: (i) Xanthi-guaninephosphoribosyltransferase gene (gpt), and (ii) phosphotransferase genome of TP5 (which meant neo). Selection using gpt is based on the ability of the enzyme encoded by homeobox gene, using xanthine as a substrate for polynucleotide synthesis; similar endogenous enzyme is absent. In a medium containing xanthine and mycophenolate acid, which blocks the conversion of inosinmonofosfata in santimonious, can survive only in cells expressing this gene gpt. Product neo blocks the inhibition of protein synthesis in eukaryotic cells by antibiotic G418 and other antibiotics in its class. Two selection procedures can be used simultaneously or sequentially to select for the expression of the genes of the immunoglobulin chains embedded in two different vector DNA in eukaryogenesis the cell.

Expressing the system

Due to inherent genetic code degeneracy other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence of the heavy and light chains, are within the scope of the present invention. The modified DNA sequence that can be used in accordance with the present invention include deletions, additions or substitutions in different nucleotide residues, giving sequences that encode the same or a functionally equivalent gene product. This gene is the first product may contain deletions, supplements or substitutions of amino acid residues in the sequence of the heavy or light chain that gives a "silent" change, producing, thus, functionally equivalent monoclonal antibody.

In accordance with the present invention the nucleotide sequence encoding the heavy and light chain of a monoclonal antibody neutralizing rabies virus, its fragment, or equivalent, is inserted into the corresponding expressing vector. This vector, which contains the necessary elements for the transcription and translation of the sequence encoding the protein is inserted so as to form a molecule of recombinant DNA that control the expression of heavy and light chain immunoglobulins for education monoclonal antibodies that neutralize the rabies virus.

The preferred recipient cell line is a myeloma cell line. Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded transfiziologii immunoglobulin genes. In addition, they have the mechanism of glycosylation of this immunoglobulin. Especially preferred recipient cell is a myeloma cell line that does not produce immunoglobulin, such as S2/0. Such cell lines produce only immunoglobulin, codere the first transfitsirovannykh immunoglobulin genes. Myeloma cells can be grown in culture or in the abdominal cavity of mice, where the secretory immunoglobulin can be obtained from ascitic fluid. Other lymphoid cells such as b cells or hybridoma cells, can serve as a suitable recipient cell.

There are several methods for transfection of lymphoid cells with vectors containing genes encoding the immunoglobulin. The preferred way of introducing DNA into lymphoid cells represent electroporation. In this method, the recipient cell is subjected to an electric pulse in the presence of this input DNA. Another way of introduction is a fusion of protoplasts. In this way lysozyme is used to remove the cell walls of bacteria carrying recombinant plasmid that contains the immunoglobulin gene. Get spheroplast merge with myeloma cells using polyethylene glycol. After fusion of protoplasts, the transfectants are selected and isolated. Another method that can be used for introducing DNA into the cells, is a calcium-phosphate precipitation.

Immunoglobulin genes can also be Express in non-lymphoid cells, such as bacterial or yeast. When they are expressed in bacteria, immunoglobulin heavy chain and the easier the e chain become part of the Taurus inclusion. Therefore, these circuits must be isolated and must be cleaned, and then assemble into functional immunoglobulin molecule. There are other ways of expression in E. coli (see, e.g., Pluckthun, A., BioTechnology 9:545-551, 1991; Skerra, A. et al., BioTechnology 9:273-278, 1991), including the secretion of E. coli in the form of a fused protein, including the signal sequence.

Example 2

Determined the complete sequences of two monoclonal antibodies against rabies virus, Mab 57 and Mab JB.1. Data monoclonal antibodies specifically associated with glycoprotein different strains of rabies virus. Postexperience treatment, and preventive treatment using a mixture of monoclonal antibodies that will neutralize the rabies virus at the site of entry of infection and prevent the spread of this virus in the Central nervous system (CNS). Thus, for transdermal or mucosal exposure to the rabies virus, a mixture of monoclonal antibodies specific to rabies, dig in to the site of the bite, and injected systemically. Because viral replication is limited almost exclusively nerve cells, neutralization and clearance of the virus by monoclonal antibodies of the present invention before entry into the CNS is effective postexplosion prevention.

A mixture of monoclonal antibodies against rabies virus injected PAC is into, who was affected or at high risk of exposure to rabies virus. A mixture of monoclonal antibodies of the present invention effectively suppresses the formation of any viral variants of rabies, which can avoid neutralization, because each monoclonal antibody in the mixture of a monoclonal antibody has specificity for the epitope, which is stored in various street rabies viruses.

The nucleotide sequence of the heavy chain Mab JB.1 person against rabies represents SEQ ID NO:9. Amino acid sequence of the heavy chain Mab JB.1 person against rabies represents SEQ ID NO:10. The nucleotide sequence of the light chain of Mab JB.1 person against rabies represents SEQ ID NO:11. Amino acid sequence of the light chain of Mab JB.1 person against rabies represents SEQ ID NO:12. The nucleotide sequence of the light chain of Mab 57 people against rabies represents SEQ ID NO:13. Amino acid sequence of the light chain of Mab 57 people against rabies represents SEQ ID NO:14. The nucleotide sequence of the heavy chain Mab 57 people against rabies represents SEQ ID NO:15. Amino acid sequence of the heavy chain Mab 57 people against rabies represents SEQ ID NO:16.

Additional examples

To ornately example 1

Neutralizing the virus effect of a monoclonal antibody having the amino acid sequence of the heavy chain SEQ ID NO:10 and amino acid sequence of the light chain of SEQ ID NO:12, tested on isolates of rabies virus below in Table 1, using the rapid fluorescent inhibition test center (RFFIT). Neutralizing activity of this antibody was compared with the activity of a commercially available preparation of human immunoglobulin against virus human (HRIG) (Imogam-rabies®, Pasteur Merieux Connaught).

The neutralizing activity of the drug antibodies were determined through repeated four double titration using cells murine neuroblastoma as a substrate for viral growth. Antibodies were diluted to 0.02 IU/ml. of 0.1 to Approximately 50 to 100 TCID50 of each virus from Table 1 were incubated with antibodies for 90 minutes at 37°C. was Added an aliquot of 200 µl of cell suspension of mouse neuroblastoma (50,000 cells/ml), and incubated for 40 hours at 37°C. After fixation with acetone virus-infected cells were visualized by staining with labeled fluoresceinisothiocyanate reagent against rabies virus. A positive assessment was based on a complete neutralization of the virus. The results are shown in Table 1 (+, neutralization; 0, no fallback).

Monoclonal the antibody SEQ ID NO:10/SEQ ID NO:12 neutralized all tested variants of the rabies virus, with the exception of one. Monoclonal antibody neutralized strain of rabies virus obtained from white-gray bat, which is not neutralized commercial preparation HRIG.

Table 1:
Neutralization of rabies virus HRIG and monoclonal antibody SEQ ID NO:10/SEQ ID NO:12
The isolate of rabies virus -HRIGMab
(1) Raccoon (Procyon lotor), Eastern part of the USA++
(2) Skunk (Mephitis mephitis), the North Central U.S.++
(3) Skunk (M. mephitis}, the South Central United States++
(4) Skunk (M. mephitis), California, USA++
(5) Gray Fox (Urocyon cinereoargenteus), Texas, US++
(6) White-gray bat (Lasiurus cinereus)0+
(7) Big brown bat (Eptesicus fuskus), new York, USA++
(8) Big brown bat (E. fuskus), Washington, USA++
(9) Silver bat (Lasionycteris noctiagans), U.S.+0
(10) the Dog, the border of the U.S.-Mexico++
(11) Dog, Thailand++
(12) the Dog, the Philippines++
(13) Arctic Fox {Alopex lagopus), Alaska, USA++

Additional example 2

Neutralizing the virus effect in vivo monoclonal antibodies with the amino acid sequence of the heavy chain SEQ ID NO:10 and amino acid sequence of the light chain of SEQ ID NO:12 were tested for isolation of rabies virus from Texas coyote as follows, using Syrian hamsters as test animals. A group of six test animals and the group of ten control animals were inoculable in the right gastrocnemius muscle 50 Microlitre 1:1000 dilution of the homogenate salivary gland coyote infected naturally with the rabies virus. Twenty-four hours hamsters were injected into the tumor inoculation of 50 μl of monoclonal antibodies, which is the dose activity, neutralizing rabies virus, approximately equal to 40 IU/kg, on each animal.

All control animals died from rabies at 16 days after infection, whereas all animals treated with a monoclonal antibody survived without clinical signs of disease.

Additional example 3

24 hours after inoculation with isolate of rabies virus from a bat of Alabama, started prophylaxis in four treatment groups of the Syrian hamster monoclonal antibody SEQ ID NO:10/SEQ ID NO:12 (40 IU/kg) or commercial HRIG (20 IU/kg), injected into a hotbed of inoculation with virus. Four groups of treatment consisted of six animals each. The control group consisted of 10 animals.

24 hours after inoculation with isolate of rabies virus from skunk North Central U.S. began prevention in four treatment groups of the Syrian hamster monoclonal antibody SEQ ID NO:10/SEQ ID NO:12 (40 IU/kg) or commercial HRIG (20 IU/kg), injected into a hotbed of inoculation with virus. Four groups of treatment consisted of six animals each. The control group consisted of 10 animals.

Ninety-five percent (19/20) of the control animals died from rabies. Sixty-seven percent (4/6) and 50% (3/6) of animals treated with 40 IU/kg monoclonal antibody SEQ ID NO:10/SEQ ID NO:12 were protected after inoculation with the virus of rabies from a bat of Alabama or the rabies virus from the skunk of the North Central U.S., respectively. For comparison, 17% (1/6) and 50% (3/6) of animals treated with 20 IU/kg HRIG, were protected after incubation with the same virus isolates.

Monoclonal antibody SEQ ID NO:10/SEQ ID NO:12 is, thus, by at least as effective as a commercial drug HRIG in protecting animals against rabies.

1. The antibody to neutralize the rabies virus containing polypeptide heavy chains, characterized by at least 80%amino acid sequence homology with SEQ ID NO:10 and a light chain polypeptide characterized by at least 80%amino acid sequence homology with SEQ ID NO:12.

2. The antibody according to claim 1, containing polypeptide heavy chains, characterized by at least 90%amino acid sequence homology with SEQ ID NO:10 and a light chain polypeptide characterized by at least 90%amino acid sequence homology with SEQ ID NO:12.

3. The antibody according to claim 1 or 2, representing the human antibody.

4. The antibody according to claim 1 or 2, representing the antibody is IgG1.

5. The antibody according to claim 2, containing polypeptide heavy chains, characterized by amino acid sequence SEQ ID NO:10 and a light chain polypeptide, characterized by amino acid sequence SEQ ID NO:12.

6. A fragment of the antibodies is a, characterized according to any one of claims 1 to 5, neutralizing rabies virus, where the specified fragment selected from the group consisting of Fv fragments, Fab fragments, f(ab')2-fragments.

7. The selected nucleic acid encoding a polypeptide containing the amino acid sequence of SEQ ID NO:10, containing a heavy chain neutralizing rabies virus antibodies.

8. Nucleic acid according to claim 7, containing the nucleotide sequence of SEQ ID NO:9, encoding the amino acid sequence of SEQ ID NO:10.

9. The selected nucleic acid encoding a polypeptide containing the amino acid sequence of SEQ ID NO:12, containing light chain neutralizing rabies virus antibodies.

10. Nucleic acid according to claim 9, containing the nucleotide sequence of SEQ ID NO:11, encoding the amino acid sequence of SEQ ID NO:12.

11. Expressing the vector containing at least one nucleic acid according to any one of claims 7 to 10.

12. The vector according to item 11, is introduced into the cell host.

13. A method of treating an individual exposed to rabies virus, introducing a specified individual a therapeutically effective amount of the antibody according to any one of claims 1 to 5 or a fragment according to claim 6.

14. The method according to item 13, where the antibody or fragment is administered to the individual in the bite or systemically.



 

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12 cl, 3 dwg, 12 ex

FIELD: microbiology, genetics of microorganisms.

SUBSTANCE: invention relates to methods for transduction of anthrax pathogen and closely related bacilli. Method for transduction of Bacillus anthracis and closely related bacilli involves incubation a mixture of bacteriophage with recipient cells taken in the definite ratio. After the first bacteriophage generation in 20 min the preparation of recipient cell receptors is added to the transducing system and the transducing system is inoculated on selective media. Invention describes a method for preparing receptors from recipient cells B. anthracis, B. thuringiensis and B. cereus. Using the proposed method provides the full-value substitution of antiphage serum in the transducing system for a cheaper and simple in preparing component, namely, the preparation prepared from recipient cell receptors that neutralizes phage effectively and prevents excessive death of the recipient strain cells in the transducing system.

EFFECT: improved method for transduction.

9 dwg, 1 tbl, 2 ex

FIELD: immunology, biology.

SUBSTANCE: invention relates to variants of IL-1β-binding molecule having common functionally active sites (CDR sites) and may bind human IL-1β. Said molecules have neutralizing activity IC50 of approximately 50 pM and binding constant KD of approximately 30 pM. Amino acid sequence is described in description of present invention. Variants of DNA constructs encoding of heavy chain and light chain of IL-1β-binding molecule are disclosed. Expression vectors carrying at least one abovementioned nuclear acid and method for production of IL-1β-binding molecules by using the same also are described.

EFFECT: IL-1β-binding molecules against human IL-1β with high neutralizing activity and binding constant useful in suppression of HAMA response.

10 cl, 1 dwg, 3 tbl, 4 ex

FIELD: biotechnology, in particular production of modified swine factor VIII (POL1212).

SUBSTANCE: DNA molecule encoding of modified swine factor VIII is cloned in expression vector, having functionality in mammalian cells. Modified swine factor VIII protein is obtained by cultivation of mammalian cell line BHK CRL-1632 (ATCC), BHK 1632, or CHO-K1, transfected with vector. Therapeutic composition for treatment of subjects suffering from deficit of factor VIII, such as haemophilia, contains effective amount of swine factor VIII protein.

EFFECT: effective agent for treatment of factor VIII deficit.

13 cl, 8 dwg, 7 ex

FIELD: biotechnology, medicine.

SUBSTANCE: invention relates to new recombinant allergens that represent mutants of allergens of the natural origin and comprising at least four mutations. Examples of recombinant allergens are allergens Bet v1 and Ves v1. The primary mutations in recombinant allergen are separated of one another by interval for at least 15 Å and is location is characterized by that at least one circle region of surface of size 800 Å doesn't comprise mutations. Recombinant allergens are used as a pharmaceutical agent as a component of pharmaceutical composition that represents vaccine against allergic response reactions. Invention describes methods for using recombinant allergens in pharmaceutical composition for producing the immune response in subject. Invention represents DNA sequences given in the invention claim that encode recombinant allergens, expressing vector comprising DNA and cell-host for providing the recombinant allergen. Also, invention describes methods for preparing pharmaceutical composition and recombinant mutant allergen. Using recombinant allergen allows decreasing the specific IgE-binding capacity as compared with IgE-binding capacity of the natural allergen. Invention can be used in medicine for preparing vaccine against allergic response reactions.

EFFECT: valuable medicinal properties of allergens.

33 cl, 62 dwg, 10 ex

FIELD: biology.

SUBSTANCE: invention relates to nucleotide sequence associated with increasing or reducing of ovulatory rate in mammalians, namely GDF-9B. Mutated GDF-9B molecule useful in modulation of ovulatory rate in female mammalians is disclosed. Also disclosed are various methods for modulation of ovulatory rate and composition for method embodiment.

EFFECT: method for inducing of sterility or reduced fertility of female mammalians.

30 cl, 14 dwg, 6 tbl

FIELD: biotechnology.

SUBSTANCE: invention relates to isolated nucleic acid sequence encoding of polypeptide with nitrilase activity, wherein nitriles are converted to carboxylic acids in presence of said nitrilase.

EFFECT: method for production of chiral carboxylic acids with high effectiveness and low cost.

10 cl, 4 dwg, 2 tbl, 1 ex

FIELD: veterinary virology.

SUBSTANCE: invention relates to 5 strains of II type porcine circovirus (PCV II) which represents causative agent of porcine post-wealing multy-systemic wasting syndrome (PMWS). Disclosed are various immunogenic compositions and vaccine based on said strains for PMWS prophylaxis and/or treatment. Also disclosed are vectors, viral preparations, cell extracts, cell culture supernatants, containing PCV II or nucleotide or protein components thereof; method for PCV II diagnosis, as well as diagnostic composition and kit.

EFFECT: new agent for treatment of porcine PMWS.

178 cl, 7 dwg, 5 tbl, 19 ex

FIELD: biotechnology, genetic engineering, virology, medicine.

SUBSTANCE: invention reports about the construction of recombinant plasmid DNAs pCL1 and pCH1 in vitro comprising artificial genes encoding light and heavy chains of human full-scale antigen against Ebola virus prepared by genetic engineering methods and created on basis of variable fragments of recombinant antibody 4d1 light and heavy chains from phage library of human single-chain antibody, and human constant genes IgG1, cytomegalovirus promoter and polyadenylation BGH site. The combining use of plasmid DNA pCL1 and pCH1 provides the biosynthesis of human recombinant full-size antibodies of class IgG1 interacting with Ebola virus. Using recombinant full-size antibodies raised against Ebola virus can be used as a basis for the development of preparations used in diagnosis and treatment of dangerous diseases caused by this infectious agent.

EFFECT: valuable medicinal properties of plasmid.

4 cl, 7 dwg, 6 ex

FIELD: immunology, biotechnology.

SUBSTANCE: invention describes antibody and its fragments neutralizing rabies virus and a method for treatment of patient subjected for effect of rabies virus by using indicated antibody and its fragment. Invention discloses variants of isolated nucleic acids encoding polypeptides carrying light and heavy chain of antibody, respectively. Also, invention describes expressing vector carrying at least one of indicated nucleic acids. Using this invention enhances span-life of patients after effect with rabies virus on them and can be used in corresponding prophylactic therapy of such patients.

EFFECT: valuable medicinal properties of antibody and nucleic acid.

14 cl, 1 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: obtained human antibody or its antigen-binding fragment specifically binds tumor necrosis factor hTNFα. The like antibodies show high affinity relative to hTNFα in vitro and in vivo. Antibodies according to the invention are taken as a full-length antibody or its antigen-binding fragment. The antibodies or their fragments are usable for detecting hTNFα and for inhibiting hTNFα activity in human beings suffering from a disorder in the case of which hTNFα activity is harmful.

EFFECT: wide range of applications of high affinity recombinant antibodies to hTNFα or their fragments of low dissociation kinetics.

15 cl, 11 dwg, 17 tbl

FIELD: biotechnology, immunology, molecular biology, medicine, pharmacy.

SUBSTANCE: invention describes the isolated human antibody or its antigen-binding fragment able to bind the human tumor necrosis factor (TNF-α). Amino acid sequence is given in the description. Invention discloses nucleic acid encoding heavy and light chain of isolated human antibody. Nucleotide sequences are given in the description. Invention describes recombinant vector expressing variable region of heavy and light chains of isolated human antibody, Chinese hamster ovary cells CHO dhfr- carrying vector. Invention discloses a method for synthesis of isolated human antibody. The isolated human antibody or its antigen-binding fragment can be used as an active component of pharmaceutical composition used in treatment of disturbances when activity of TNF-α is harmful. Using the invention allows neutralization of effect of TNF-α in case when its activity is harmful. Invention can be used in medicine.

EFFECT: valuable medicinal properties of antibody, improved method for synthesis.

17 cl, 11 dwg, 17 tbl, 4 ex

FIELD: immunology; treatment of mediated diseases IL-1 and failures.

SUBSTANCE: bonding molecule IL-1β which is antibody to human IL-1β and especially human antibody to human IL-1β where hypervariable sections CDRs of heavy and light chains have definite amino acid sequences. Antibody may be used for treatment of mediated disease IL-1, for example osteoarthritis, osteoporosis and other inflammatory processes of bones of rheumatism or podagra nature. Constructions of deoxyribonucleic acid are described which code heavy and light chains or their fragments and expressive vectors which may be replicated in cells including deoxyribonucleic acid constructions. Method of obtaining bonding molecule IL-1β by means of cell transformed by vector is described. Proposed antibody may be used both in prophylactic and treatment of diseases.

EFFECT: enhanced efficiency.

15 cl, 3 dwg, 5 ex

FIELD: medicine, immunobiology, pharmacy.

SUBSTANCE: humanized monoclonal antibody (monAb) or its fragments comprises heavy and/or light chain with the binding rate constant with AILIM 1.0 x 103 (1/M x s) and above, and the dissociation rate constant between monAb and AILIM 1.0 x 10-3 (1/s) or less. MonAb shows also a nucleotide sequence encoding variable region of light and/or heavy chain and corresponding amino acid sequences. Invention relates to DNA and it part encoding monAb or its fragments, and vectors comprising nucleotide sequences encoding antibody or its fragments. The humanized monAb can be prepared by using a genetically recombinant host. MonAb is comprised as a component of pharmaceutical compositions used for inhibition or induction of AILIM-mediated transfer of signal into cell for induction of antibody-dependent cytotoxicity against AILIM-expressing cell and others. Invention can be effective in treatment of different autoimmune diseases associated with AILIM-mediated transfer of co-stimulating signal. Invention can be used in medicine for treatment of diseases associated with AILIM-mediated transfer of co-stimulating signal.

EFFECT: valuable medicinal properties of antibody.

75 cl, 78 dwg, 14 ex

FIELD: genetic engineering, molecular biology.

SUBSTANCE: invention proposes a method for detecting genes encoding membrane-bound transmembrane proteins. Method involves expression of the nucleic acid chimeric sequence in the cell-host consisting of DNA fragment encoding secreting protein that is able to bind antigen and DNA fragment to be tested; interaction of cells expressing the fused protein with antigen; selection of cells on surface of that indicated antigen is bound; isolation of recombinant vector containing in selected cells of DNA fragment to be tested and, if necessary, determination of its sequence. Also, invention proposes the developed vector constructions and comprising their sets designated for realization of the proposed method. Invention provides significant simplifying the screening process of libraries and cloning genes encoding transmembrane proteins. Invention can be used for detecting and preparing genes encoding any membrane-bound proteins used in different branches of science and practice.

EFFECT: improved isolating method, valuable biological properties of protein.

27 cl, 7 dwg, 1 tbl, 8 ex

FIELD: genetic engineering, immunology, medicine.

SUBSTANCE: invention relates to new antibodies directed against antigenic complex CD3 and can be used in therapeutic aims. Antibody IgG elicits the affinity binding with respect to antigenic complex CD3 wherein heavy chain comprises skeleton of the human variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 2, 4 and 6 and their corresponding conservatively modified variants. Light chain comprises skeleton of the rodent variable region in common with at least one CD3 taken among amino acid sequences SEQ ID NO 8, 10 and 12 and their corresponding conservatively modified variants. Antibody is prepared by culturing procaryotic or eucaryotic cell co-transformed with vector comprising recombinant nucleic acid that encodes antibody light chain and vector comprising recombinant nucleic acid that encodes antibody heavy chain. Antibody is administrated in the patient suffering with malignant tumor or needing in immunosuppression in the effective dose. Invention provides preparing chimeric antibodies against CD3 that are produced by expression systems of procaryotic and eucaryotic cells with the enhanced yield.

EFFECT: improved preparing methods, valuable medicinal properties of antibody.

33 cl, 5 dwg, 1 ex

The invention relates to biotechnology, in particular to the immunoglobulin E (IgE), IgE antagonists, anti-IgE antibodies capable of binding to human IgE, and to a method of improving polypeptides, including anti-IgE antibodies

The invention relates to the field of biotechnology and medicine, namely, to new sequences of DNA nucleotides and amino acids sequences of monoclonal antibodies (MABS) generated against lymphoblastoid cells, and peptides that bind MAT

FIELD: biotechnology, hybridoma technology.

SUBSTANCE: hybridoma T2/S-6E11 is prepared by fusion of murine myeloma strain Sp-2/0 and murine BALB/c splenocytes immunized with the TOPC virus, strain CoD, purified preparation inactivated with concentrate formaldehyde solution and by using polyethylene glycol-1000 Da and the following cloning by method of limited dilution. Prepared hybridoma T2/S-6E11 produces monoclonal antibodies (MCAb) to epitopes of the abovementioned pathogen. Specificity of prepared MCAb is shown by absence of cross-reactions in IFA-test with Hantaan and Ebol viruses and with noninfected substrates accumulated by TOPC virus. IFA-test system based on MCAb provides carrying out the specific detection of TOPC virus in analyzed samples. The sensitivity of this test-system based on MCAb is estimated to be 2.5 x 103 plaque-forming units (virus) in cubic centimeter (PFU/cm3). Using the invention in immunological researches in creature of diagnosticum used for detection of TOPC virus in samples provides enhancing the specificity of analysis in detection of coronaviruses.

EFFECT: valuable properties of strain.

3 tbl, 1 ex

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