SU1742326A1 - Strain of hybridous cultured mammalian cells mus musculus l - a producer of monoclonal antibodies to mycobacterium bovis - Google Patents

Abstract

Uses: biotechnology, medicine, veterinarian. SUMMARY OF THE INVENTION: A strain is obtained by hybridization of Balb / c mice immunized with a suspension of M.bovis strains inactivated by gamma irradiation, isolated from sick cows, with PzO myeloma cells. The secretion of monoclonal antibodies on days 3–4 of cultivation is 40–50 µg / ml and 5–7 mg / ml after 20–25 days in ascitic fluid. The strain deposited under the number VSKK (P) No. 506D. Monoclonal antibodies can be used to identify microbacteria. 1 tab. with C

Description

This invention relates to biotechnology and is concerned with the production of monoclonal antibodies (MCAT) for bovine-type mycobacterium tuberculosis (M. bovis) and can be used in medicine, in particular phthisiology, and also in veterinary medicine.

The aim of the invention is to obtain a strain of hybrid cultured mouse Mus musculusi cells producing MCAT to intact M. bovis. The strain obtained by hybridization of cells of the spleen of mice of the VA line in / s. immunized with gamma irradiation inactivated by field strains of M.bovis, with Rz01 myeloma cells in a ratio of 3: 1 using 50% polyethylene glycol (mol.m. 3550). Cell mixture is poured into 96-well

plates of 2-10 splenocytes per well. For cultivation and breeding of hybrids, RPM1-1640 medium containing 20% calf fetal serum, M hypoxanthine M, M aminopterin and 1, M thymidine is used. Hybrid cell culture fluid was tested for the content of antibodies specifically binding to intact M.bovJs in an enzyme-linked immunosorbent assay (ELISA). As a result, hybridoma 2A1 producing antibodies highly specific for M. bovis was selected from 1000 cell populations. Hybridoma cells are cloned twice by the method of limiting dilutions of 1 cell. per well, containing 4-10 cells as a substrate. spleen. After the second cloning, 99% of the positive clones were detected.

Antibody production is maintained for 8 passages in cell culture.

A portion of the 2A1 hybridoma cells is frozen in fetal bovine serum with the addition of 10% DMSO in liquid nitrogen for storage. Another part is used to obtain the tumor in ascites form in BALB / c mice by intraperitoneal injection of 4-106 cells. animals prepared by intraperitoneal injection of 0.5 ml vaseline oil for 3 to 30 days before injection of cells. Ascites occurs after 20-25 days. Immunoglobulins were isolated from ascitic fluid using sodium sulfate (2.53 M solution).

Strain 2A1 is stored in the Specialized Collection of transplanted /, somatic cells of the vertebrates of the Institute of Cytology of the Academy of Sciences of the USSR under the number 506 D and is characterized by the following features. Cultivation conditions. RPM1-1640 medium with 20% fetal bovine serum, 4 mM L-glutimine, 10 mM sodium pyruvate, amino acids for the basal Eagle's medium, vitamins for the Eagle's medium, 0.05 mM mercaptoethanol, and in an atmosphere of 5% carbon dioxide.

Plastic dishes are used to grow the strain. Sowed dose of 100 thousand, cells / ml. The cells are passaged once every 3-4 days, the multiplicity of sieving is 1:10. Suspension culture.

Cultivation in animals.

BALB / c mice are suitable for growing tumors. Fresh mice 3 to 30 days before the injection of the strain are injected intraperitoneally with 0.5 ml of pristine or petroleum jelly. The strain is injected intraperitoneally at 4 - 5 106 cells. among the Needle. Ascites occurs after 20-25 days. Strain is not intertwined. Strain productivity: secretion of monoclonal antibodies on days 3–4 of cultivation 40–50 µg in 1 ml of culture medium and 5–7 mg in 1 ml of ascitic fluid as determined by radioimmunoadsorption inhibition.

Characteristic of a useful product: monoclonal antibodies possess selective specificity for surface antigens of mycobacterium tuberculosis of bovine species. Stable secretion of the product is maintained for at least 10 passages in vitro and when cells are cultured in mice in the form of an ascites tumor.

Contamination with bacteria, fungi, mycoplasma was not detected.

Cryoconservation: 3.5 - 4.0-10 cells. strain canned in 1.0 ml of tel

fetal serum with the addition of 10% dimethyl sulfoxide in plastic ampoules. The freezing is carried out in the program freezer according to the following

the program: the temperature is reduced to 4 ° C in 1 minute to 4 ° C and 1 ° C in each minute to -40 ° K. Cells are stored in liquid nitrogen. Defrosting: quickly at 37 ° C. The viability after defrosting on the inclusion of tri0 pan blue is 80-85%.

Example: Hybridoma cells stored in liquid nitrogen are thawed rapidly at 37 ° C, washed with medium 199, and seeded into culture medium, which consists of RPM1-1640 supplemented with 20% fetal bovine serum, 4 mM L-glutamine , 10 mM sodium pyruvate, 0.05 mM mercaptoethanol and grown at 37 ° C in an atmosphere of 5% carbon dioxide. By sev10 dose 105 cl. in 1 ml. After 3-4 days, the concentration of antibodies in the culture fluid reaches 40 - 50 µg / ml.

The specificity of the interaction of antibodies with mycobacterium tuberculosis bovine

5 species are detected by a solid phase immunoassay method. For this, a polystyrene 96-well microtitre panel is sorbed by those killed by gamma irradiation or chloroform mycobacterium: 0.1 mg of culture per well in 100 μl of carbonate-bicarbonate buffer (0.2 M; pH 9.6) at 37 ° C over night After saturation of the panel with 10% solution of egg protein in phosphate-buffered saline

5 (0.02 M; pH 7.2) to reduce the nonspecific binding of antibodies with plastic, 100 µl of the culture medium of strain 506D was added to the panel cells for 1 h, after which the panel was washed three times with tap water and then with water with 0, 05% Tween 20 and, finally, a conjugate of rabbit antibodies against mouse immunoglobulins covalent5 but associated with peroxidase dissolved in but-saline buffer from 10% egg protein and 0.05% Tween 20 introduced (for 1 hour at 37 ° C ).

After the completion of the incubation, the unbound reaction products are washed in this way, and the resulting peroxidase,

0 and therefore antibodies against mycobacterium tuberculosis, are determined by the cleavage of the substrate (NaOa) in the presence of the chromogen, orthophenylene diamine.

The results of enzyme immunoassay5 for the specificity of MCAT 506D with mycobacteria are presented in the table.

The results presented in the table indicate that the obtained MCAT specifically react with mycobacteria of bovine tuberculosis.

Claims (1)

Claims of the invention: strain of hybrid cultured animal cells Mus musculus L VSCC (P) No. 506 D-producer of monoclonal antibodies to Mucobacterlum bovls. Mycobacterium M.bovls BCG (vaccine) M.bovls BCG {Omsk VNIIBTZH) M.bovis Bov nus-8 (MNIIT) M.bovls BovinuS-8 (OMCKHu VNIIBTZH) M.bovis (strains derived from patients with tuberculosis of cows) M. tuberculosls (strains derived from people with tuberculosis,) Atypical mycobacteria: M.kansasli M.fortultum, M.lntracellulare M.gastrll M.phley M.avlum Mdeggae. i M.martnum M.smegmatls .; Optical density index 0.59 ± 0.01 0.65 ± 0.01 1.19 ± 0.01 0.55 ± 0.01 0.63 ± 0.03 0.04 ± 0.01 0.06 ± 0.005 0.06 ± 0.01 0.04 ± 0.01 0.04 ± 0.01 0.04 ± 0.01 0.04 ± 0.01 0.03 ± 0.01 0.05 ± 0 , 01 0.06 + 0.01