Strain of hybrid cultivated murine cells mus musculus-6f3 producing monoclonal antibody nontolerant to hemagglutinin protein of high-pathogen strain virus a/duck/novosibirsk/56/05 h5n1

FIELD: medicine.

SUBSTANCE: invention aims at preparation of new strain of hybrid cells Mus. Musculus 6F3 - a producer of monoclonal antibody (MCA) to hemagglutinin protein of high-pathogen avian influenza virus A/duck/Novosibirsk/56/05. Strain 6F3 is prepared by fusing murine myeloma cells Sp2/0 with murine spleen cells BALB/c, immunised with a purified and inactivated preparation of avian influenza virus A/H5N1 (strain A/duck/Novosibirsk/56/05). Hybridoma produced MCA belong to IgA class. Strain 6F3 is deposited in the Collection of cell culture of Ivanovsky State Research Institution of Virology of the Russian Academy of Medical Sciences, No. 8/2/3. Using hybridoma allows producing specific monoclonal antibodies to hemagglutinin protein of avian influenza virus A/H5N1.

EFFECT: possibility to use antibodies to studying the antigenic structure of hemagglutinin for differential diagnostics of avian influenza virus A/H5 serotype.

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The invention relates to the field of biotechnology and Virology and the receipt of a new strain of hybrid cells Mus. Musculus 6F3 - producer of monoclonal antibodies to protein hemagglutinin of the virus of avian influenza A(H5N1), which can be used to make diagnostic and preventive medicines.

In recent years, the virus of avian influenza A(H5N1) virus caused a major epidemic among poultry in Southeast Asia, as well as outbreaks in poultry in other regions of the world. However, some variants of H5N1 viruses were able to infect humans, causing severe disease, often fatal. According to who, in the world registered 373 cases of human infection with the H5N1 virus, 236 people died [1]. On the territory of the Russian Federation (RF) avian viruses of serotypes H5N2 and H5N3 were isolated from wild and domestic birds [2]. Most of the viruses was characterized by weak pathogenicity. In 2005, for the first time on the territory of the Russian Federation isolates were highly pathogenic variants of influenza A(H5N1). All the strains isolated in the Novosibirsk region, studied in detail in a State institution research Institute of Virology. Dijanoveckog Russian Academy of medical Sciences (Institute of Virology. Dijanoveckog RAMS), two of them were deposited in the State collection of viruses GUNII Virology. DI Ivanovo, RAMS, and highly productive strain A/duck/Novosibirsk/56/05 H5N1 patented (EN 2309983, priority 25.11.2005) [3, 4]. In 2006-2007 were deposited in the STB 16 strains of influenza virus H5N1, selected as in the Asian and European parts of the Russian Federation [5,6]. Phylogenetic analysis of the genome of these strains have established their belonging to the highly pathogenic avian viruses Qinghai-Siberian genotype [7].

Strains of avian viruses circulating in South-East Asia, is well studied [8-10]. Analysis of the RNA genome, antigenic and three-dimensional structure of the hemagglutinin H5 revealed significant differences between laboratorni strain A/duck/Singapore/3/97 and selected later from humans by highly pathogenic strain A/Vietnam/1203/04 [11]. This indicates the need to control for evolutionary changes in the virus of avian influenza H5 to improve diagnostic tests and the development of adequate vaccines.

One of the experimental tools for the analysis of the antigenic structure and properties of viral proteins are monoclonal antibodies (MAB). A new and promising direction in biotechnology is to create humanized antibodies that can be used for the prevention and treatment of humans. Based on murine MCA by genetic engineering are designed antibodies mouse-man, Fab-fragments which contain hyperv realnye areas of specific immunoglobulins mouse µa, having neutralizing activity, and Fc-fragments (constant part) derived from human immunoglobulins. As humanized antibodies contain the minimum number of fragments of mouse antibodies, they can be used for the prevention and treatment of humans. So, B.J.Hanson et al. on the basis of two µa to influenza A/H5N1 virus (strains A/Vietnam/I 203/04 and a/HK/213/03) were constructed humanized antibodies with neutralizing activity was preserved. The prophylactic and therapeutic effect of humanized antibodies was investigated in mice: different amounts of the MAB was administered before or after infection with a lethal dose of influenza A/H5N1. It was found that both the MCA were effective in the prevention scheme; in the medical scheme one of the antibodies were more active and showed complete protection from the disease at a concentration of 1 mg/kg of body weight [9]. Thus, passive immunization can be an alternative strategy for the prevention and treatment of pandemic variants of influenza A/H5N1 human. Most valuable are the "secretory antibodies of the IgA class, which is suitable for prophylactic intranasal [12]. Thus it is shown that protection from influenza a significant role to play secretory IgA antibodies detected in the nasopharynx: they are the first special is practical barrier to the virus, spreads through airborne droplets [13, 14].

Well-known foreign collection murine MCA to the virus of avian influenza A/H5N1 produced by strains of hybrid animal cells [15,16]. Produced by these hybridomas µa belong to the IgG class.

Analogues of hybrid strains of animal cells Mus. Musculus - producers µa to highly pathogenic virus of avian influenza A/H5N1 virus circulating on the territory of the Russian Federation, is unknown.

The essence of the invention consists of creating hybridoma producing MCA, immunoreactive with the protein hemagglutinin of highly pathogenic virus of avian influenza strain A/duck/Novosibirsk/56/05 H5N1 and related to the class IgA, and used for diagnostics and prevention.

The essential features of the invention, coinciding with the characteristics of the prototype include the following :

1. The strain of hybrid cells 6F3 producing monoclonal antibodies to the hemagglutinin of the virus of avian influenza A/H5, obtained by the method of hybridoma technology by immunization of BALB/c mice.

2. MCA 6F3 interact with homologous virus in the biological and immunochemical reactions.

The essential features of the invention, which differs from the characteristics of the prototype are as follows.

1. The strain of hybrid cells 6F3 produces monoclonal antibodies specific for immunoglobulin A (IgA), whereas in FR the types µa belong to the IgG class.

2. Immunized animals was the preparation of the virus of avian influenza A/H5N1 virus (strain A/duck/Novosibirsk/56/05), allocated on the territory of the Russian Federation, which refers to highly pathogenic viruses of avian influenza A/H5N1 Qinghai-Siberian genotype that is different from the virus of avian influenza A/H5N1 virus used in the prototypes.

3. For immunization of animals used purified and inactivated preparation of influenza virus in birds, whereas prototypes mice were immunized with the recombinant protein hemagglutinin or DNA plasmid that encodes a protein hemagglutinin.

The technical result of the claimed invention to provide a strain of hybrid cells 6F3, producing μa to the virus of avian influenza A/H5N1 virus (strain A/duck/Novosibirsk/56/05)allocated in the Novosibirsk region in 2005 (EN 2309983, priority 25.11.2005) [4].

This technical result is achieved by hybridization of cultured murine myeloma cells Sp2/0 with spleen cells of mice BALB/c mice immunized with purified, and inaktivirovannye preparation of the virus of avian influenza A/H5N1 virus (strain A/duck/Novosibirsk/56/05), which was obtained by cultivation and purification of the virus.

The inventive strain of hybrid cells Mus. Musculus 6F3 obtained in a State institution research Institute of Virology. Dijanoveckog Russian Academy of medical Sciences and deposited in the Collection of purevia is by cell line genetics, Institute of Virology. Dijanoveckog RAMS number 8/2/3. Author name hybridoma cell line - 6F3.

The Strain Of Mus. Musculus 6F3 characterized by the following features:

Pedigree strain. The hybrid strain of cultured cells of Mus. Musculus 6F3 was obtained by fusion of mouse myeloma Sp2/0 cells in the spleen of mice BALB/c mice immunized with purified, and inaktivirovannye preparation of the virus of avian influenza A/H5N1 virus (strain A/duck/Novosibirsk/56/05). As a melting agent used reagent PEG-DMSO company ("Hibri-Max", "Sigma", USA). Hybrid cells were cultured on selective medium GAT, then double-cloned by the method of limiting dilutions, using as cells kormeluk peritoneal macrophages of BALB/c mice.

The number of passages by the time the Deposit was 8-12.

Contamination by bacteria and fungi were not found.

Morphological features. Cell culture consists of weakly attached to the substrate rounded cells of various sizes with large nuclei.

Cultural properties. As the growth media for culturing used the medium RPMI-1640 containing 20% fetal calf serum (ETS), 4.5 g/l glucose, 3 mm glutamine, and 0.2 U/ml insulin, 50 μg/ml gentamicin. Character growth is stationary suspension, the method for removing the shaking. The frequency of passage - 2-3 days. Sowing dose of 200 thousand cells in 1 ml of radnoti sieving - 1:2-1:3.

The cultivation of hybridoma in the body of the animal. Female BALB/c mice (the vivarium of the Institute of Virology. Dijanoveckog RAMS) previously injected intraperitoneally 0.5 ml of piers ("Sigma", USA). Through 14-21 day animals vaccinated 107/ml hybrid cells into the abdominal cavity. After 7-14 days formed ascitic tumor. From one animal you can get 3-5 ml of ascitic fluid containing MAB. Hybridoma inoculated in 100% of cases.

Cryopreservation of hybridoma. 24 hours before freezing hybridoma subcultured 1:2. Precipitated by centrifugation (1000 rpm, 10 min) cells suspended in createsite environment (90% ETS, 10% DMSO) and poured into vials at a concentration of 3-5 million/ml 1-1,8 ml. Stored 24 hours at-70C°, then transferred to liquid nitrogen.

Characterization of immunoglobulins. The definition of the class of immunoglobulin produced µa 6F3, was performed by ELISA using a set of "Mouse-Hybridoma-Subtyping Kit" (Boehringer Mannheim, Germany). In hole panel barbirolli purified preparation of the virus of avian influenza A/duck/Novosibirsk/56/05 at a concentration of 2 μg/ml in phosphate-buffered saline (FSB), and incubated with MCA in serial dilutions. After washing was added conjugates - antimurine antibodies to different classes (IgG, IgA, IgM) and subclasses (IgGI, IgG2a, IgG2b, IgG3)antibodies labeled with horseradish peroxidase. As the substrate used tetramethylbenzidine. Pok is connected, that produced by hybridomas 6F3 µa interacted only with conjugates against IgA (optical density of more than 1.0 for all dilutions used ICA), which allowed to refer them to the IgA class.

The method of obtaining the claimed strain.

The immunization. Female mice of BALB/c derived from kennel "Pushchino (Moscow region), were immunized intraperitoneally 4 times with an interval of 2 weeks at a dose of 100 μg of antigen/mouse. For the first immunization, the antigen was injected in a mixture with complete adjuvant-blockers, for the following three - mixed with incomplete adjuvant's adjuvant. Booster dose (100 μg of antigen in saline, 0.1 ml) was administered intravenously 3 days before the selection of the spleen.

The merge. To use merge cells of mouse spleen cells and mouse myeloma Sp2/0 in the ratio of 1:5. The mixture of cells centrifuged, the supernatant carefully removed and the cell precipitate was added 1 ml of PEG-DMSO, consisting of 45% of Page with molecular weight of 1450 and 10% DMSO, ("Hybri-Max", "Sigma", USA). After 3 min pause the mixture was centrifuged 15 min at 1000 Rev/min the Precipitate washed twice with medium without serum and is dissolved in the growth medium. Cells are distributed in 96-well plates ("Costar, USA) in 100 μl into the hole. Selection of hybrid cells is carried out in the environment GAT ("Sigma", USA), consisting of a nutrient medium RPMI-1640, which added 20% fetal calf is La serum ("Gibco", USA), 0.1 mm gipoksantina, 0.04 mm thymidine and 0.01 mm aminopterin.

The selection of specific hybrids was performed using enzyme-linked immunosorbent assay (ELISA) and response inhibition of haemagglutination (rtga). As AG in both reactions used homologous virus A/duck/Novosibirsk/56/05.

Example 1. The determination of the activity of MCA 6F3 using enzyme-linked immunosorbent assay. 96-hole panel (NUNC, Denmark) were senzibilizirani purified preparation of influenza virus A/duck/Novosibirsk/56/05 at a concentration of 2 μg/ml, were incubated with MCA in serial dilutions. After the washing was added to the conjugate - antimurine antibodies labeled with horseradish peroxidase (DAKO, Denmark). As the substrate used tetramethylbenzidine (Sigma, USA). It was shown that the titer of the MCA was 1/107.

Example 2. The determination of the activity of MCA 6F3 in response inhibition of haemagglutination (rtga). Rtha was performed using human red blood cells I (0) group by standard methods [17]. Dose of influenza viruses of humans and animals was 8 agglutinating units. It was found that MCA 6F3 inhibit hemagglutination of the virus of avian influenza strain A/duck/Novosibirsk/56/05 in breeding 1/10240. The obtained data are given in the Table.

Example 3. The specificity of ICA 6F3 against proteins of the virus of avian influenza A/H5N1 strain A/duck/Novosibirsk/56/05, was determined by the method of Western blot test. Proteins which were separated in 10% polyacrylamide gel and transferred by the method electroelution on the nitrocellulose with a pore diameter of 0.45 μm ("Bio-Rad", USA). Incubation with MCA was carried out for 1 h or 18 h on a shaker at room temperature. The result showed that the processing strips of nitrocellulose AT to mouse Ig labeled with peroxidase (Sigma, USA). As a negative control was used S2/0 - ascitic fluid at a dilution of 1/500, as a positive control serum of mice used to get a hybrid in a dilution of 1/500. These experiments showed that ICA 6F3 interact with the heavy chain of the hemagglutinin HA1 (Mm 57 kD).

Example 4. Neutralizing activity µa 6F3 studied in reactions of biological neutralization. MCA warmed up at 56°C for 30 min in a water bath. Serial dilution of the MAB were incubated with influenza a viruses (infectious multiplicity 100 TCD50) for 2 h at 37°C and were made in cell culture spew or dog kidney (MDCK). The results were taken into account through 72-96 hours, when in the infected culture, not treated µa, developed maximum cytopathic effect (JRS) [18]. It is established that the MCA 6F3 neutralized infectious activity of the virus of avian influenza strain A/duck/Novosibirsk/56/05, breeding 1/5120. The obtained data are given in the Table.

Example 5. The study of the ability of the ICA 6F3 to identify infected cells in the reaction of indirect immunofluorescence (nIf). Cells spew infected with a virus (A/duck/Novosibirsk/56/05 with infections the ion plurality of 10-100 TCD 50. 24 hours after infection the cells were fixed with chilled acetone for 30 minutes On fixed preparations were applied µa 6F3 and incubated for 1 hour at 37°C. Then washed with running water and layered antimycin serum, labeled FITZ (DAKO, Denmark). It is shown that ICA 6F3 were able to paint cells spew infected with homologous virus of avian influenza A/duck/Novosibirsk/56/05. Coloring viral hypertension was detected in the cytoplasm of infected cells.

Example 6. The study of the ability of the ICA 6F3 to inhibit the hemagglutination and neutralize infectious activity of different influenza viruses in birds and humans. To analyze the properties of the ICA used 7 strains of the virus of avian influenza serotype A/H5: pathogenic strains of avian influenza A(H5N1)isolated in Russia: A/duck/Novosibirsk/56/05, A/duck/Tuva/01/06, A/chicken/Moscow/2/07; A/chicken /Krasnodar/329/07, antigen And/H5 from a diagnostic kit who 2003-2004 (H5 influenza kit VA 2710, Atlanta, USA); the strains of A(H5N3): reference virus strain of avian influenza A/tern/South Africa/61 and A/duck/Primorie/2633/01. In addition, used the avian viruses of other serotypes: A/duck/Czechoslovakia/56 (H4N6)A/turkey/Massachusetts/65 (H6N2)A/turkey/Wisconsin/66 (H9N2), and the virus of human influenza A/New Caledonia/20/99 (H1N1). The results of rcga and reactions of biological neutralization (RBN) is presented in the Table. MCA 6F3 showed the greatest activity with all 7 of the studied strains of the virus g is IPPA birds A/H5. When studying MCA 6F3 with other serotypes of influenza a (H1, H4, h6, H9), the reaction was negative.

These properties of strain hybrid cells 8/2/3 (author's name cell lines 6F3) allows us to conclude that for the first time on the basis of mouse myeloma received hybridoma Mus. Musculus 6F3 - producer µa to protein hemagglutinin of highly pathogenic virus of avian influenza A/H5N1 strain A/duck/Novosibirsk/56/05. The virus was isolated on the territory of the Russian Federation in the Novosibirsk region in 2005 and applies to "Qingdao" branch. The strain of hybrid cells allows to obtain murine immunoglobulin IgA in the amount of 2-5 mg purified antibodies from ml of ascitic fluid. MCA specific react with the virus of avian influenza A/H5N1 strain A/duck/Novosibirsk/56/05, ELISA (titer was at least 1/107). MCA with high activity inhibit hemagglutination of all the studied viruses of avian influenza serotype A/H5 circulating on the territory of the Russian Federation and the reference strains obtained from the who. This allows their use for investigation of thin antigenic structure and epitope specificity of new virus isolates of avian influenza A/H5, and also for the differential diagnosis of avian viruses of serotype A/H5. Activity µa 6F3 in the reaction nIf provides the ability to use them for detection of the virus in smears and fingerprints tissue of patients and infected birds, animals of man. MCA 6F3 with high neutralizing activity and producing secretory antibodies of the IgA class, can be "humanitarian" and used for prophylactic intranasal introduction of the people.

The above properties of the strain of Mus. Musculus 6F3 distinguish it from all previously described hybridomas producing µa to protein hemagglutinin of the virus of avian influenza A/H5N1.

The list of references

1. Cumulative Number of Confirmed Human Cases of Avian Influenza A/(H5N1) Reported to WHO. 18 March 2008.(http://www.who.int/en).

2. Lions D.K., Amnicola S., Fedyakin ETC etc. Ecology and evolution of influenza viruses in Russia (1979-2002,) / / Matters. Virusol. - 2004. - T. 49, No. 3. - P.17-24.

3. Lions D.K., Saltanov M., Deryabin p. g and others Isolation of strains of influenza virus A/H5N1 from poultry and wild birds during the period of the epidemic in Western Siberia (July 2005) and their deposition in the state collection of viruses (08 August, 2005) // Matters. Virusol. - 2006. - So 51, No. 1. - C.11-14.

4. Method the primary isolation of influenza virus strains And the virus strain A/Duck/Novosibirsk/56/05 H5N1 for the preparation of diagnostic, prophylactic and therapeutic drugs, to assess the antiviral activity of various compounds.// Patent RU 2 309 983, priority 25.11.2005.

5. Lions D.K., Prilipov A.G., Saltanov APPLICANT and other Molecular genetic analysis of the biological properties of highly pathogenic strains of influenza A/H5N1 virus isolated from wild and up to asnic birds during the epizootic in Western Siberia (July 2005) // Matters. Virusol. - 2006. - So 51, No. 2. - P.15-19.

6. Lions D.K., Saltanov M., Deryabin p. g and others Isolation of highly pathogenic (HPAI) strains of influenza virus A/H5N1 from wild birds in the hearth of the epidemic on the lake UBS-Nur (June 2006) and their deposition in the State collection of viruses RF (3 July 2006) // Matters. Virusol. - 2006. - So 51, No. 6. - P.14-16.

7. Lions D.K., Saltanov M., Prilipov A.G. and other Molecular diagnostic characteristics of strain A/chicken/Moscow/2/2007 (H5N1) outbreak of epizootic of highly pathogenic influenza a among agricultural birds in the suburbs (February 2007) // Matters. Virusol. - 2007. - T. 52, No. 6. - P.40-47.

8. Chen H., Smith, G. J. D., K. S. Li et al. Establishment of multiple sublineages of H5N1 influenza virus in Asia: Implications for pandemic control. // Proc. Natl. Acad. Sci. - 2006. - 103 - P.2845-2850.

9. B.J. Hanson, Boon A.C.M., Lim APC et al. Passive immunoprophylaxis and therapy with humanized monoclonal antibody specific for influenza A H5 hemagglutinin in mice. // Respiratory Research. - 2006. - V.7. - N1. - P.126-136.

10.Peiris J.S.M., de Jong MD, Guan Y. Avian Influenza Virus (H5N1): a Threat to Human Health.// Clin.Microbiol.Rev. - 2007. - V. 20. - N2. - P. 243-267.

11. Webster, R.G., E.A. Govorkova H5N1 influenza-continuing evolution and spread. // N.Engl.J.Med. - 2006. - V. 355. - N21. - P. 2174-2177.

12. Renegar K.B., Jackson G.D., Mestecky J. In vitro comparison of the biologic activities of monomeric monoclonal IgA, polymeric IgA, and secretory IgA. // J.Immunol. - 1998. - V.160. - N3. - P.1219-1223.

13. Brokstad, K., SOH R., J. Olofsson et al. Parental influenza vaccination dosage a rapid systemic and local immune response. // J Inf. Dis. - 1995. - V.171. - P.198-203.

14. Ghendon Y. The immune response to influenza infection. // Acta virol. - 1990. - V.34. - P.295-304.

15. He Q., Velumani, S., Du Q., Lim, C.W., Ng, F.K., R. Donis, Kwang J. Detection of H5 Avian Infuenza Virusesby Antigen-Capture Enzyme-Linked Immunosorbent Assay Using H5-Specific Monoclonal Antibody. Clin vaccine and immunology, 2007, p.617-623.

16. Horimoto T., Fukuda n, Iwatsuki-Horimito K., Guan Y., Lim W, Peiris m, Sugii, S., Odagiri T., Tashiro T., Kawaoka Y. Antigenic Differences between H5N1 Human Influenza Viruses Isolated in 1997 and 2003. J.Vet.Med.Sci., 2004, 66(3), p.303-305.

17. Deryabin p. g, Butenko A.M., Burtseva H. haemagglutination Reaction and the reaction of inhibition of haemagglutination. // In Medical Virology" under the editorship of Academician RAMS D.K. Lvov. Moscow. MYTH - 2008. - S.312-318.

18. Deryabin p. g, Butenko A.M. Reaction of biological neutralization. // In Medical Virology" under the editorship of Academician RAMS Dkilla. Moscow. MYTH - 2008. - S-310.

The hybrid strain of cultured mouse cells Mus. Musculus - 6F3, deposited in the collection of transplantable cell cultures at the Institute of Virology. Dijanoveckog RAMS number 8/2/3 producing monoclonal antibody immunoreactive with the protein hemagglutinin of highly pathogenic virus strain A/duck/Novosibirsk/56/05 H5N1 related to the IgA class.



 

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FIELD: agriculture.

SUBSTANCE: invention relates to the field of genetic engineering and cloning. Transgenic cow is disclosed, having low activity of prion protein as a result of one or several genetically designed mutations. Specified transgenic cows are also genetically modified for expression of xenoantibodies.

EFFECT: as a result of their resistance to prion diseases, such as trembling disease of cattle, such cows are a safe source of human antibodies for use in pharmaceutics and a safe source of agricultural products.

12 cl, 112 dwg, 13 tbl, 19 ex

FIELD: medicine.

SUBSTANCE: invention refers to antibody specifically getting bound with PRO87299 version. In addition, the antibody according to the invention has ability to block interaction HVEM and PRO87299 and to function as PRO87299 agonist. The antibody of agonist nature is produced by hybridoma Btig5F5.1 or Btig3B1.9. For the antibody, there is established amino acid sequence given in the description. The invention discloses the methods of using the antibodies to stimulate or reduction of immune response in immune-associated diseases connected, to relieve lymphoma, and inflammatory disease in requiring mammal, to detect polypeptide PRO87299 in a sample and to manage rejection of grafted cells.

EFFECT: antibody is an immunomodulator that allows applying therapeutically identical medicinal agents both to intensify and reduce immune response.

16 cl, 34 dwg, 7 tbl, 20 ex

FIELD: chemistry.

SUBSTANCE: proposed is a chimeric or humanised monoclonal antibody against hepatocyte growth factor, produced from L2G7 antibody. Invented is a mouse antibody L2G7, produced by hybridoma ATCC PTA-5162, and the said hydbridoma. Described is a cell line, producing a chimeric or humanised monoclonal antibody against hepatocyte growth factor. Proposed is a pharmaceutical composition and a method of treating tumours based on the said antibody.

EFFECT: use of the invention provides for a neutralising antibody against hepatocyte growth factor, which can be used in treating human cancer.

7 cl, 12 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention concerns area of medicine and concerns the therapeutic agent for treatment of diseases related to children's chronic arthritic diseases, for example, actually children's chronic arthritic diseases, Still's disease and similar to it, including an antagonist of the interleukin-6 IL-6 receptor as an active ingredient, humanised antibody PM-1. Advantage of the invention consists in efficiency increase.

EFFECT: efficiency increase.

5 cl, 4 ex

FIELD: biotechnology, immunology.

SUBSTANCE: disclosed are variants of chimerical anti-IL-6 antibodies based on mice CLB-8 antibody. Each antibody contains constant region from one or more human antibodies. Described are variants of nuclear acids encoding anti-IL-6 antibody, vectors and host cells. Developed is method for production of anti-IL-6 antibody by using nuclear acid or vector. Described are variants of composition for application in method for modulation of malignant disease or immune disorder mediated with IL-6. Developed is method for treatment or modulation of malignant disease or immune disorder mediated with IL-6.

EFFECT: variant of chimerical anti-IL-6 antibody with high affinity of mice anti-IL-6 antibody and reduced immonogenicity.

26 cl, 16 dwg, 1 tbl, 8 ex

FIELD: medicine, biotechnology.

SUBSTANCE: invention proposes variants of antibodies showing specificity to peptide domain located by both side of hinged site R76S77 in pro-BNP(1-108). Indicated antibodies recognize specifically also circulating pro-BNP(1-108) in human serum or plasma samples but they don't recognize practically peptides BNP(1-76) or BNP(77-108). Also, invention describes variants of peptides used in preparing antibodies. Amino acid sequence is given in the invention description. Also, invention discloses methods for preparing indicated antibodies and among of them by using indicated peptides. Also, invention describes methods for preparing antibody-secreting hybridoma, and hybridoma is disclosed prepared by indicated method. Also, invention describes a monoclonal antibody secreted by hybridoma 3D4 and deposited at number CNCM I-3073. Also, invention discloses variants for diagnosis of cardiac insufficiency in vitro and by using antibodies proposed by the invention. Also, invention describes a set used for detecting pro-BNP(1-108) in a biological sample. Using this invention simplifies detection of pro-BNP(1-108) circulating in human serum or plasma samples and provides specific detection of pro-BNP(1-108) that can be used in early diagnosis of human cardiac insufficiency.

EFFECT: valuable medicinal properties of antibodies.

24 cl, 16 dwg, 5 tbl, 20 ex

FIELD: medicine, analytical immunology.

SUBSTANCE: invention relates to a set of reagents used in quantitative determination of secretory immunoglobulin A (sIgA) that provides assaying status of topical immunity and immunodeficient states in carrying out analysis of serum and secrets of human body. Proposed set comprises a plate with immobilized monoclonal antibodies, five calibrating samples containing 0; 1.0; 5.0; 10.0 or 20.0 mcg of sIgA/ml, conjugate of murine monoclonal antibodies with horse radish peroxidase and a reagent for carrying out the enzymatic reaction. Murine monoclonal antibodies produced by hybrid strain of animal Mus musculus L., № PKKK (P) 676D cultured cells, are immobilized on a carrier. Conjugate comprises murine monoclonal antibodies produced by another strain - Mus musculus L., № PKKK (P) 677D. The advantage of invention involves enhancing sensitivity in assay of sIgA.

EFFECT: improved assay method.

3 tbl, 5 ex

The invention relates to medicine, in particular to the treatment and pulmonology, and for the treatment of acute lung injury and fibrosis

FIELD: biotechnology, in particular GHRH-fixing protein.

SUBSTANCE: disclosed is new protein having molecular mass of about 90.9 kDa which includes fragments of identified peptide sequences 1, 2 and 3. Method for production of said protein includes extraction of Pilocarpus heterophyllus plant cells with water, vacuum filtering, deposition of extracted proteins, centrifugation, and gel filtering of obtained filtrate. Also produced is monoclonal antibody which specifically fixes isolated protein.

EFFECT: agent for producing of effective drugs and pharmaceutical composition against GHRH action and treatment of proliferative diseases.

12 cl, 5 dwg, 2 ex

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