RU1794949C - Strain of hydrous cultured cells mus musculus l - a producer of monoclonal antibodies to mycobacteria antigen of human type mycobacterium tuberculosis @@@ rv of molecular mass 20 kda - Google Patents

Abstract

Usage: biotechnology, immunologists, medicine. SUMMARY OF THE INVENTION: a strain is obtained by splenocyte fusion of BALB / c mice immunized with M sonicates, tuberculosis HsvRV, with mouse cells of the X63-Ad653 line of myeloma using a 50% solution of polyethylene glycol 6000 with a 3: 1 ratio of spleen cells to myeloma cells. The strain is designated MT-1 and deposited under the number VSCK (P) 488D. The strain produces monoclonal antibodies directed to the human mycobacterium antigen mol.m. 20 kDa. The antibody titer reaches 1: 256 in the culture fluid, and 1: 25x103 in the ascitic fluid. 1 tab.

Description

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WITH

The invention relates to medicine, namely to immunology. The invention describes the preparation and properties of a hybrid clone of MT-1 transplantable mouse cells (hybridoma) producing monoclonal antibodies to human mycobacteria M. tuberculosls HayRV type.

The preparation of this producer opens up prospects for improving the methods for serodiagnosis of tuberculosis, namely, the determination of antitucurculous antibodies and antigens of mycobacterium tuberculosis in serum and other biological fluids of a person by enzyme immunoassay, radioimmunological and immunofluorescence analysis.

Numerous data are available on the preparation of hybridomas producing monoclonal antibodies to bacteria. However, the present invention is characterized in that the resulting hybridoma MT-1 produces monoclonal antibodies (MKATs) against M. tuberculosls HavRV.

McAb for mycobacterium tuberculosis of human type was obtained. The analog strain differs from the claimed strain by its specificity: directivity to another antigenic determinant, namely, 20 kDa.

The purpose of the invention is the production of Mabs that specifically react with human mycobacteria of the M. tuberculosis type H37RV.

The goal is achieved by creating a strain of hybrid cells by fusion of murine myeloma cells with spleen cells of mice immunized with M.tub sonicum. HayRV, capable of growing in mice as an ascites tumor and producing ascites in high titer.

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ABOUT

clonal antibodies to the human mycobacterium antigen (M.tuberculosls

H37RV).

The strain of cells MT-1 was deposited in the All-Union Collection of Cell Cultures of the Institute of Cytology of the Academy of Sciences of the USSR in Leningrad under no.

A method for producing a strain. BALB / c mice were immunized subcutaneously at b points with 100 μg M.tub sonicate. in incomplete Freud's adjuvant twice, with an interval of 2 weeks. A booster dose of 40 μg of sonicate was administered intravenously three days before sampling spleen cells for hybridization. The titer of antibodies specific for HsyRV sonicate in the blood serum of an immune mouse was determined by enzyme-linked immunosorbent assay with the antigens that were immunized and amounted to 1: 16 thousand hours. Fusion of splenocytes with X63-Ad8653 mouse murine myeloma cells was performed using a 50% PEG (polyethylene glycol) 6000 (Merck) solution at a 3: 1 ratio of spleen cells to myeloma cells. After the fusion procedure, the cells were suspended in GAT selective medium at the rate of 3 × 10 4 cells / well of a 96-well panel. 7 days after hybridization, the panels were viewed using an inverted microscope. Hybrid clone growth was observed in 80% of the wells. By enzyme-linked immunosorbent assay (ELISA) after 14-17 days. Tested the culture fluid from the wells with growing clones for the presence of specific antibodies. A set of sonic antigens from M.tub was used to select ELISA hybrid clones. HayRV, BCG, Intracellulare, Scrofulaceum, Fortuitum and E.coll.

Cultural characteristics of the line.

Culture medium: RPM1-1640 medium with 10% fetal bovine serum, 1 mM sodium pyruvate, 10 mM HEPES buffer, 2 mM L-glutamine, 5-10 5 M 2-mercaptoethanol, 50 u / ml gentamicin.

Growth pattern: stationary suspension

Method of removal: shaking Passaging frequency: 2-3 days Seeding dose: 2-3-10 cells / ml Seeding rate: 1: 2-1: 3 Preservation of cells: cells are frozen after 1,3.10,15 and 25 passages in culture and after growth in the form of an ascites tumor in mice. The total number of ampoules is 30, 4-5 million cells per ampoule. Cryoprotective medium: embryonic calf serum with 10-20% dimethyl sulfoxide. Mode

freezing: cells in a cryoprotective medium were stored for 1-3 days at -70 ° C, after which they were placed in liquid nitrogen. Defrost survival - 80%,

Information about the contaminants of the line: bacteria - no, fungi - no, mycoplasma - no,

yeast - no.

Culture morphologists: the culture consists of cells that weakly adhere to the substrate with the usual morphology for hybrid antibody-forming cells (round cells with large nuclei and a thin rim of the cytoplasm, large nucleoli, 1-2 in each nucleus).

Kariologists of culture: the karyotype corresponds to the species Mus.musculus, the Modal number of chromosomes is 89, the range of variation is from 78 to 92 chromosomes.

Example 1. In vitro cultivation of hybrid cells at a planting concentration of 200 thousand cells / ml of growth medium, the cell culture is incubated for 2-3 days in a stationary state at 37 ° C in an atmosphere of 5% COa. Cell concentration in excess of 1

ppm is not favorable for cell growth and causes their death. The culture fluid from the 3-day culture vial is tested for the presence of monoclonal antibodies,

PRI me R 2. In vivo cultivation of hybrid cells: cultured cells in vitro at a concentration not exceeding 500 thousand / ml, i.e. in the logarithmic phase of growth, centrifuged

u1 at 200d. The cell pellet after centrifugation was suspended and the number of viable cells was counted with trypan blue. 5-7 million cells / ml are introduced into the abdominal cavity of BALB / c mice,

pre-treated marinas, the treatment is carried out once 1-3 weeks before the introduction of hybridoma cells by injection of 0.5 ml pristan. After 1-3 weeks (depending on the number of injected

cells) ascites tumors form. The amount of ascites fluid accumulating in the abdominal cavity of mice is on average 5 ml.

PRI me R 3. The activity and specificity of monoclonal antibodies in ELISA. In order to carry out the immunochemical characterization of the obtained McAb from the supernatants of the culture of MT-1 hybridoma and ascites fluid, a globulin fraction was isolated

by drying (MnfZO at 50% saturation. In the globulin fraction, the amount of protein was determined spectrophotometrically at a wavelength of 260 and 280 nm (when diluting globulin 50 times).

The specificity characterization of the obtained Mabs was performed using ELISA. For analysis, the same set of sonic antigens was used, on which supernatants were screened for selection of clones. The antigen concentration was 10 μg / ml. The globulin fraction of McAt was used in the following concentrations: 10, 2.5, 1.25, 0.625 µg / ml. 25 μl of globulins of McAb in triplicates for each antigen were applied to an antigens sensitized and blocked 1% bovine serum albumin plate. The reaction was exhibited by second antibodies against mouse immunoglobulins labeled with horseradish peroxidase. As a substrate, 30% NaOa and orthophenylenediamine were used. The average optical density of triplicates at a wavelength of 492 nm is presented in the table.

Claims (1)

SUMMARY OF THE INVENTION Strain of hybrid cultured Mus musculus L cells. HSCK (P) No. 488D is a producer of monoclonal antibodies to anti-MkAt Mt-1 specificity characteristics. in reaction with sonicates mycobacteria in ELISA 0 5 Based on the analysis performed, it was shown that MkAt MT-1 binds to M. tuberculosis sonicatum at a high level, while M. BCG sonicate was weaker, and with other sonicates, the binding was at the level of the reaction background. The use of the obtained antibodies will make it possible to increase the efficiency of tuberculosis diagnostics by increasing the specificity of immunological reactions in the determination of anti-tuberculosis antibodies and antigens of human type mycobacteria in patients with tuberculosis, and will also enable early detection of mycobacterium tuberculosis infection during preventive examination of people. twenty the gene of mycobacteria of the human type Mycobacterlum tuberculosis HayRV with mol.m, 20 kDa.