Strain 1h8 of permanent hybridomal line of cells of mouse mus. musculus - producent of monoclonal antibodies to igg of sheep

FIELD: veterinary.

SUBSTANCE: obtained is strain 1H8 of hybridomal line of cells of mouse Mus. musculus - producent of monoclonal antibodies to IgG of sheep, suitable for biotechnology in elaboration of diagnostic preparations. Strain is deposited with Special Collection of re-inoculated somatic cell cultures of agricultural and commerciall sold animals by No 73. When determined by method of immuno-enzymatic analysys (IEA) antibodies titers in native culture liquid constituted in IEA 1:32-1:64, in ascitic liquid 1:640-1:5120. Monoclonal antibodies are specific to immunoglobulin IgG of sheep and do not react with immunoglobulin of cattle. When used for fixation on solid phase of glycoproteidal antigen of cattle (C) leucosis virus (in composition of complex glycoproteidal antigen- monoclonal antibodies of sheep to glycoproteidal antigen) in IEA, they ensure strength of fixation and optimal availability of antigen for antibodies in testes samples.

EFFECT: strain 1H8 can be used in production of immuno-enzymatic test-system for diagnostics of C leucosis, which will allow to increase efficiency of sanitation measures, reduce terms of enhancement of adverse in terms of leucosis cattle-breeding farms.

1 tbl, 4 ex

 

The present invention relates to the field of veterinary biotechnology, namely, to obtain strains of hybrid cells producing monoclonal antibodies that can be used for the preparation of highly specific diagnostic reagents for detection of antibodies against various infectious agents, in particular leukosis virus of cattle.

The leukosis virus in cattle causes malignant lymphoproliferative disease of cattle - leucosis, which ranks first in the structure of infectious diseases of cattle in the Russian Federation. The infectious process is characterized by the absence of viremia with simultaneous production of antibodies to proteins of the virus, which are dominated by antibodies to glycoprotein viral envelope (gp51). For diagnosis of the disease are widely used serological methods, namely the reaction diffusion precipitation in agar gel and enzyme-linked immunosorbent assay.

The need for this work due to the fact that the greatest sensitivity and specificity in detecting antibodies to the virus leukemia have test systems, which are based on the principle of the enzyme immunoassay. The use of monoclonal antibodies for the preparation of peroxidase labeled species-specific anti the l to the immunoglobulin IgG cattle can improve the specificity of the assay. Part of a diagnostic test-systems includes the antigen leukosis virus, which is obtained from the culture fluid transplantable cell cultures, particularly cultures of kidney cells of the embryo sheep chronically infected with the virus leukemia. The use of monoclonal antibodies for immobilization of the antigen on the plastic tablet titration eliminates time-consuming and often inefficient purification of the antigen and to significantly improve the specificity and sensitivity of the analysis. As we developed a test system designed to detect antibodies to the virus leukemia cattle in biological fluids of cattle, for immobilization of the antigen used monoclonal antibodies sheep Ovis aries to glikoproteinom antigen leukosis virus produced interspecific hybrid line S. The necessity of using monoclonal antibodies to immunoglobulins IgG sheep as antibodies, fixing complex glycoproteins antigen - monoclonal antibodies to glikoproteinom antigen on the solid phase, due to the fact that the cultivation of interspecific hybrid lines IS possible only in vitro, but not in animals. However, the antibody in the culture fluid is much lower than in ascitic, and, in addition, the nutrient medium composition for culturing posto is the R cell cultures included serum (10-20%), which pollutes the preparation of monoclonal antibodies. This has necessitated the use for fixing antigen monoclonal antibodies to immunoglobulin IgG sheep with no cross-reactivity with immunoglobulins of cattle.

In the available literature information about the strains of hybridoma cell cultures producing monoclonal antibodies to immunoglobulin IgG sheep not found.

The objective of our research was to obtain a strain of hybrid cells with in vitro high production of monoclonal antibodies to immunoglobulin IgG sheep, do not give cross-reactions with antibodies cattle.

The proposed method of obtaining strain is as follows.

The strain of hybrid cells obtained by fusion of cells of transplantable culture of mouse myeloma P3-X63-Ag 8.653 with lymphocytes of mouse spleen of BALB/c immunized twice by injection of 100 μg of drug immunoglobulin IgG sheep, purified chromatographic methods.

At the height of the immune response (3 days after last injection) conducted a fusion of splenocytes of immunized mice with cells of the mouse myeloma line P3-X63-Ag 8.653.

The cell fusion was performed by standard methods using PEG with 1500 mm. The ratio of myeloma cells and lymphocytes was 1:4-1:10. Breeding hybrid p is bodily on the environment, containing gipoksantin, aminopterin and thymidine. Hybridoma were cultured on 96-well panels manufactured by "Nunc" in the medium RPMI-1640 with the addition of 10% serum of cow embryos in an atmosphere containing 5% CO2.

The screening of hybridomas for the production of specific monoclonal antibodies was performed by the method of solid-phase ELISA using purified preparations of IgG sheep and cattle for sensitization of the solid phase and conjugate polyclonal antibodies to immunoglobulin IgG mouse peroxidase for detection of bound peroxidase antibodies. Cloning and reklamiranje cell cultures were performed on 96-well panels method of limiting dilutions using macrophage feeder layer.

The result was selected and propagated clone of cells IN producing monoclonal antibodies to immunoglobulin IgG sheep, do not give cross-reactions with antibodies in cattle. This clone of cells was serially propagated in 96-, 24-hole panels, and then in the culture mattresses.

The culture fluid was examined on the content of specific antibodies. The titer of specific antibodies in ELISA was 1:32-1:64. Preparations of monoclonal antibodies was obtained by double precipitation with a solution of ammonium sulfate to 50% saturation with subsequent dialysis. Was defined species, DVD is a possibility and specificity of monoclonal antibodies ELISA methods.

The proposed strain has the following properties.

Morphological features. The cells are uniform, round shape with a large oval nucleus containing from 1 to 3, 1-2, nucleoli. When seeding the cells are evenly distributed on the surface of the substrate and in the culture fluid. The proliferation index is equal to 5-6.

Cultural properties. Cells were cultured in medium RPMI-1640 with the addition of 10% serum of cow embryos. Cells grow in suspension and partially attached to the surface of the culture vessel. The seeding concentration of 1×104cells/ml in 5-7 days after sowing 80% of the culture fluid is removed along with her cells. The remaining cells add the same volume of fresh medium. pH of 7.2 to 7.4. The frequency of subculturing - 1 every 3-5 days. The multiplicity of sieving 1:5.

Cultivation in animals. Cells cause the formation of ascites in line BALB/c mice treated with dock for 7-10 days prior to intraperitoneal administration of 2-10×106cells/mouse. Activity in ELISA ascitic fluid - 1:640-1:5120.

Immunological properties. The strain produces mouse monoclonal antibodies against immunoglobulin IgG sheep having no cross-reactivity with immunoglobulins of cattle. The antibody specificity is determined by the method immunofermentnom what about the analysis in tablets, sensitized immunoglobulin IgG sheep and cattle. In the tablet make the culture fluid and incubated 1.5 hours at 37°C, making the conjugate antibodies against immunoglobulin IgG mouse peroxidase, incubated in the same mode. After each step, conduct laundering holes from unbound reagents. Make the substrate mixture. The intensity of staining appreciate a 5-10 min. Values of optical density in the wells sensitized immunoglobulin IgG sheep and cattle, respectively, more than 1.5 and less than 0.05.

The definition of cultural activity, ascitic fluid conducting enzyme-linked immunosorbent assay in tablets, sensitised immunoglobulin IgG sheep. Prepare a twofold serial dilution of the material researched and analyzed similarly to the specificity study. As a control, use the serum intact linear BALB/c mice at a dilution of 1:25. For the title take the greatest dilution of the investigated material, for which the optical density at two or more times greater than the optical density in the control.

The contamination. Contamination protozoa, fungi, bacteria, mycoplasmas, viruses are not detected.

Storage of cultures. Environment for freezing: 70% RPMI-1640 medium, 20% whey of cow embryos, 10% Dima is insulted. The concentration of cells in plastic cryovials of 1.5×106cells/ml. Mode freeze: up to -70°C, 1°/min, followed by liquid nitrogen (-196°C). Recovery after freezing: rapid thawing at 37°C, centrifuged at 1200 rpm for 10 min, re-suspension in the growth medium. Viability after thawing 70-80%.

The strain used in the manufacture of a diagnostic test system for the detection of antibodies to the virus leukemia cattle enzyme-linked immunosorbent assay.

Advantages: antibodies to immunoglobulin IgG sheep, do not give cross-reactions with antibodies cattle.

The proposed strain N hybrid cell line mouse Mus. musculus producing monoclonal mouse antibodies to immunoglobulin IgG sheep - deposited in a "special Collection transplantable somatic cell cultures of agricultural and game animals" (she RAAS) under No. 73.

The strain of hybrid cells N is a permanent cell line suitable for biotechnology in producing diagnostic products.

Example 1. Immunological properties of monoclonal antibodies. The specificity of the antibodies in the culture fluid determine enzyme-linked immunosorbent assay in tablets, sensitised immunoglobulin IgG sheep and cattle according to the above method. The values of optical density in the wells sensitized immunoglobulin IgG sheep and cattle, respectively 1,750 and to 0.032. The ratio of the values of optical density in excess of 54, suggests that the monoclonal antibodies react with immunoglobulins sheep, but do not react with immunoglobulins of cattle.

Example 2. Cultivation of the strain in vitro and quantifying the production of antibodies. Cells of strain N cultivated in glass mattresses at a temperature 37,0°C in a nutrient medium consisting of RPMI-1640 medium and 10% serum embryos blood of antibiotics (penicillin and streptomycin at 500000 unit/l). The optimum planting density of 2×104cells/ml of medium. In 2-3 days after the formation of the suspension culture fluid partially remove and add the required amount of fresh nutrient medium. The titer of antibodies to immunoglobulin IgG sheep in the culture fluid was in ELISA 1:64.

Example 3. Cultivation in animals and quantifying the production of antibodies. Cells of strain at a dose of 2-10×106cells/mouse in 0.5 ml of phosphate-saline buffer was injected into the abdominal cavity linear BALB/c mice, which previously 7 days introduced 0.3 ml of the Wharf. 10-14 days received ascitic fluid, the titer of specific monoclonal antibodies to IgG OVC is which amounted in ELISA 1:2560.

Example 4. Conducting enzyme immunoassay using monoclonal antibodies. Drugs monoclonal antibodies were obtained from ascitic fluid three-precipitation of a solution of ammonium sulfate to 50% saturation with subsequent dialysis. Later they were used as antibodies, fixing monoclonal antibodies sheep to glikoproteinom antigen gp51 VLCRC S, in the enzyme-linked immunosorbent assay for detecting antibodies to the virus leukemia cattle in biological fluids of cattle. Immobilization of the antigen on the micropanel conducted in three stages.

In the first stage, the surface of the plastic panel for micrometrology was senzibilizirani due to nonspecific adsorption of the monoclonal antibody to sheep immunoglobulins produced by strain N (deposited in she RAAS under No. 73) from a solution with a concentration of 5 µg/ml at pH of 7.2-2-7,4. Incubation was carried out for 16-20 hours at 4°C. In the second stage, conducted immobilization due to specific immunological interaction of monoclonal antibodies to glikoproteinom antigen gp51 VLCRC from the culture fluid of strain S (deposited in she RAAS under No. 72) for 1.5 hours at 37°C. In the third stage was performed immobilization also due to specific immunological interaction of antigen leukosis virus to the total cattle from the culture fluid virusprobleme culture. Excess reagents after each stage of sensitization of the solid phase and conducting immunoassay washed with a solution of detergent in phosphate-saline buffer solution. Made test and analyze samples of blood serum of cattle and incubated for 1.5 hours at 37°C. as the detecting antibody used peroxidase labeled monoclonal antibodies against globulin cattle, do not give cross-reactions with antibodies sheep produced by strain A (deposited in she RAAS under No. 71). Incubation was carried out for 1.5 hours at 37°C. the reaction Accounting conducted by the change in optical density of samples compared to control samples after addition of the substrate mixture containing tetramethylbenzidine (TMB) and hydrogen peroxide H2About2. Positive thought of the sample, the optical density in which two or more times greater than the optical density of the negative control.

The study 662 blood sera from disadvantaged by the leukemia economy in parallel in the reaction diffusion precipitation (RDP) and enzyme-linked immunosorbent analysis results (RDP+/ELISA+; RDP-/ELISA) was noted in 560, or 84.6%of the mismatch (API+/ELISA; RDP-/ELISA+) - 15.4% of cases. Only 3 positive on financial p the tats RDP samples reacted negative in ELISA (0.5 percent). In ELISA were identified 190 (28,7%) positive samples, whereas in the RDP - 94 (14,2%), i.e., 14.5%, or twice more. The sensitivity of enzyme immunoassay surpassed RDP with glikoproteinami antigen and did not yield to her specificity.

The proposed strain tested with positive results in laboratory conditions SSI-Russian Institute of experimental veterinary medicine named. Arrowlink and Kursk bio from 2006 to 2007

The feasibility study. The resulting strain IN hybrid cells producing monoclonal antibodies to immunoglobulin IgG sheep, characterized by the following useful properties:

- is a permanent cell line suitable for biotechnology in producing diagnostic products;

- has a high level of production of monoclonal antibodies. When determining enzyme-linked immunosorbent assay antibody titers in native culture fluid was in ELISA 1:32-1:64, ascitic fluid 1:640-1:512;

- monoconal antibodies specific for immunoglobulin IgG sheep;

- monoclonal antibodies do not react with immunoglobulins of cattle;

- monoclonal antibodies when used for fixing on solid phase glikoproteinov antigen leukosis virus in cattle in the complex glycoproteins antigen - monoclonal antibodies sheep to glikoproteinom antigen) immunoassay analysis provide the strength of fixation and optimal availability of antigen for antibodies in the test sample.

Strain IN producing monoclonal antibody to sheep IgG - will find application in the manufacture of enzyme immunoassay system for the diagnosis of leukemia in cattle. Using this test system will improve the effectiveness of the Wellness interventions, to reduce the duration of recovery affected with leukemia livestock farms and eventually dramatically reduce the incidence of bovine leukemia.

Sources of information

"Immunofluorescence and related staining techniques" Ed. by W. Knapp. Elsevier: North-Holland Biomedical Press. - 1978. - 215-221.

Strain IN continuous hybridoma cell line mouse Mus. Musculus producing monoclonal antibody to sheep IgG deposited in a "special Collection transplantable somatic cell cultures of agricultural and game animals (she RAAS)" Moscow under No. 73.



 

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1 tbl, 4 ex

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1 tbl, 4 ex

FIELD: medicine.

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2 ex

FIELD: biology.

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53 cl, 4 tbl, 10 dwg, 2 ex

FIELD: biotechnology, immunology.

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EFFECT: valuable biological and medicinal properties of antibody and hybridoma.

8 cl, 7 dwg, 8 ex

FIELD: microbiological and immunological methods.

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2 cl, 6 dwg

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

FIELD: biotechnology, virology.

SUBSTANCE: invention relates to preparing a new strain of hybrid cells of Mus musculus L., NIIMB-280 (9E2), as a producer of monoclonal antibodies to the West Nile virus (WNV) protein E. West Nile virus (strain WNV/LEIV-VIg99-27889) is isolated in Volgograd district in 1999 year from a patient. Producing monoclonal antibodies can be used effectively for detection of the strain WNV/LEIV-VIg99-27880 of WNV that causes human diseases in Russia territory. New hybrid strain of cells is obtained by fusion of murine myeloma cells p3-X63/Ag8.653 (NS0/1) with murine splenocytes BALB/c immunized with the purified and inactivated preparation WNV (strain WNV/LEIV-VIg99-27889). The strain of hybrid cells Mus musculus L., NIIMB-280 (9E2), is deposited in Collection of cellular cultures of NII cellular cultures GNTS VB "VEKTOR" at number № NIIMB-280. Author's name of hybridoma cellular strain is 9E2. Using hybridoma allows preparing specific monoclonal antibodies raised to the West Nile virus protein E that, in turn, gives a possibility for identification of WNV and to standardize the content of protein E in immunodiagnostics.

EFFECT: valuable properties of strain.

1 dwg, 3 ex

FIELD: biotechnology, hybridoma technology.

SUBSTANCE: hybridoma strain is prepared by fusion of murine plasmocytoma Sp2/0-Ag.8 and B-lymphocytes of murine spleen of the inbred strain BALB/c immunized with protein-polysaccharide complex from Y. enterocolitica. Hybridoma produces monoclonal antibodies of isotype IgG to Y. enterocolitica O3 and O9 serovars used as components of IFA-test-system for identification of indicated serovars that are isolated most often in European areas from sick humans, agricultural animals and from objects of environment. The usage of monoclonal antibodies producing by hybridoma allows carrying out the identification of Y. enterocolitica strains of indicated serovars representing the most epidemic danger among other intestine-persistent microorganisms. Invention can be used in the development of diagnostic test-systems for identification of Y. enterocolitica strains O3 and O9 serovars for aims laboratory diagnosis in the public health, veterinary science and in carrying out scientific investigations.

EFFECT: valuable properties of strain.

1 tbl, 2 ex

FIELD: biology, biotechnology, medicine.

SUBSTANCE: strain of murine hybridoma cells is prepared by fusion of murine splenocytes immunized with cell lysates of lymphoblastoid line RAMOS with cells of murine myeloma. Hybridoma secrets monoclonal antibodies directed to antigen of molecular mass 110 kDa located on microtubules in cell cytoplasm and detected in nuclear cells of different species. Using the invention allows studying pathogenesis of cell dividing and state of mitotic activity of health and malignant cells, and evaluation of anti-mitotic (anti-tumor) effect of different chemopreparations and substances. Invention can be used for identifying phase of mitotic activity of cells.

EFFECT: valuable properties of strain.

2 dwg

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