SU1640157A1 - Strain of hybridous cultivating mammalian cells, mus musculus l, used for preparation of monoclonal antibodies for dna-dependent rna-polymerase of bacteriophage t7 - Google Patents

Description

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(21) 4685570/13

(22) 04/25/89

(46) 04/07/91. Bul № 13

(71) Institute of Molecular Biology. V.A. Engelhardt

(72) I.L. Degt roar, D.A. Kostyuk and S.N. Kochetkov

(53) 578.085.23 (088.8)

(56) Carroll S.B., Stollar B.D. Proc. Nat. Acad. Sci. USA 1982, v. 79, p. 7233-7237.

(54) MUSCULUS L MUSCULUS L HYBRIDAL CULTIVATED ANTIBODY CELLS, USED TO RECEIVE MONOCLONAL ANTIBODIES TO DNA-DEPENDENT RNA GOLYMERASE BACTERIOPHAG T7

(57) The invention relates to hybridoma technology and can be used to improve the quality of the secreted eukaryotic RNA polymerase enzyme. The aim of the invention is to obtain a hybridoma strain producing monoclonal antibodies.

(Мрн AT) and RNA polymerase of the T7 bacteriophage. The strain is obtained by hybridization of splenocytes of BALB / C mice immunized with the purified preparation of the bacteriophage T7 RNA polymerase with cells of the mouse myeloma of the line X63, AG 8.653. Cells were cultured in DMEM medium with fetal bovine serum (10%), glutamine (2 mmol), sodium pyruvate (1 mmol), 2-mercaptoethanol (0.05 mmol), penicillin / streptomycin (1000 units / ml). The frequency of passaging 2-3 times a week, sowing dose of 150 thousand cells per 1 ml, the multiplicity of seeding 5-10 times. The productivity of strain 10 (by ELISA) in culture medium and 3 x 105 in ascitic fluid. My ATs secreted by the strain specifically interact with the R7 polymerase of bacteriophage T7. Strain deposited under

BCKK (II) -351D. Mon AT refers t-s to IgG1 class.

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The invention relates to a hybrid technology and can be used to improve the quality of the secreted zukaryotic RNA polymerase enzyme.

The aim of the invention is to obtain a hybridoma strain producing monoclonal antibodies (My AT) to bacteriophage T7 RNA polymerase.

Strain was prepared as follows.

Eight-week-old BALB / C mice are immunized in the hind feet with 35 µg of antigen each with a full anti-

Juvant Freund. After 4 weeks Mice are immunized subcutaneously in several places along the back, neck, and body. About 50 μg of antigen in Freund's incomplete adjuvant. After 4 weeks 50 µg of antigen is administered intraperitoneally without adjuvant. After 10 days, non-immune BaLB / C mice lethal irradiated (750 rad) on an x-ray machine, 24 hours after irradiation, lymphocytes obtained from the spleen of an immune mouse were injected intravenously. Simultaneously, mice are teased with 30 μg of antigen.

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tricylobe. Four days later, after lymphocyte transplantation, fusion is put

The titer of antibodies specific to the bacteriophage T7 DNA-dependent RNA polymerase in the serum of a test mouse is 1–2000, as determined using enzyme-linked immunosorbent assay.

The fusion of splenocytes with cells of the murine mienoma of line X63.AH8.653 is performed using PEG with a molar of 4000. The ratio of the number of myeloma cells to the number of splenocytes is 1: 1. After fusion, the cells are introduced into the wells of 96-well panels. 1 day before fusion, splenic cells were planted in wells of 96-well panels (1 mln cells / ml 100 μl per well) Selection of hybridoma clones was performed on GAT medium prepared in DMEM supplemented with 20% embryonic bodies Whose serum and 40 mcg / ml antibiotic gentamicin. Cells are cultured in an incubator containing CO, in the atmosphere. Hybrid cell clones appear after a week in 60% of the wells.

After 14-17 days, culture fluids from the wells with clones are tested for the presence of specific antibodies by the indirect solid phase enzyme immunoassay (TIFA) method.

The cloning of positive cultures was carried out by limiting dilution, depositing 0.5 cells per well of a 96-well plate with a nourishing layer of splenic cells. The HMB-4F7 clone was selected as the best producer of monoclonal antibodies and re-cloned according to the same scheme as for the first time. One of the reclones, characterized by maximum activity and the formation of ascitic tumors in 100% of the BALB / C vaccinated mice, is called IMB-4P7.

The cell strain HMB-4F7 was deposited in the All-Union Cell Culture Collection of the Institute of Cytology, USSR Academy of Sciences under the number BGKK (II) 35ID.

The strain is characterized by the following features.

Morphological signs. Suspension culture consisting of large, rounded cells with large dramas and a thin rim of cytoplasm. The nucleoli are large, 1-2 in the core.

Cultural features. Cultivation media - Wednesday AFTER: syvoro

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cow embryo (10%), neonatal serum (10%), glutamine (2 mmol), sodium pyruvate (1 mmol), 2-mercaptoethanol (0.05 mmol) ,. penicillin / streptomycin (1000 U / ml). The growth pattern is a stationary suspension. Shooting method - shaking. Passing frequency 2-3 times a week. Sown dose of 150 thousand cells per 1 ml. The multiplicity of sieving 5-10 times.

Control contamination. Bacteria, fungi and mycoplasmas are not detected.

The culture is grafted and grows in the form of ascites in the peritoneal cavity of BALB / C mice. 7 days prior to the introduction of the cells, recipient mice were given 0.5 ml of pristane. Ascites is formed 10–15 days after administration of 1–5 million gels.

The productivity of the strain. Primary culture is monoclonal. Two cloning was performed. All clones are positive. For 30 passages of the IMB-4P7 clone, the intensity of antibody production did not change (the antibody titer by ELISA in culture / culture medium 10, and in typical liquid 3x10).

My ATs belong to the class G10 immunoglobulins.

Cell preservation. IMB-4R7 cells were frozen at the 36th passage. The total number of ampoules is 20, 4 million cells per ampoule. The cryoprotective medium is a growth medium with 50-60% fetal calf surami and TO% dimethyl sulfoxide. Survival rate when defrosting 95%.

Example,. Cultivation of hybridoma cells IMB-4R7.

Hybrid cells are introduced into cell culture vials at a concentration of 100–200 thousand cells in 1 ml of DMEM medium containing 10% fetal cow, 10% serum of newborn calves, 2 mmol

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glutamine, 1 mmol pyruvate sodium, 0.05 mmol 2-mercaptoethanol and 1000 units. penicillin / streptomycin. The cells are incubated for 2-3 days in a steady state at 37 ° C in an atmosphere of 6% COa. A cell concentration in excess of 1 ppm in 1 ml is unfavorable for cell growth and causes their death. The culture fluid from the three-day culture vials was tested for the presence of Mont AT. When seeding, the cells are shaken, diluted with growth medium to the indicated concentration, and poured into vials.

Example 2: Cultivation of hybridoma cells IMB-4P7. . Cells growing in culture flasks, 1-2 days after subculture, centrifuged for 10 minutes at 800 rpm. Cells should be in a concentration not exceeding 500 thousand / ml, i.e. be in the logarithmic growth phase. After the centrifugation, the cell pellet is resuspended in a certain volume of culture medium without serum and the number of viable cells is counted using trypan blue. The concentration of cells is adjusted to 1-10 mln / ml and injected into the abdominal cavity of BALB / C mice that have been pretreated with a pier. The treatment is carried out once for 1-3 weeks before the introduction of hybridoma cells by injection of 0.5 ml of bailiff. After 1-3 weeks (depending on the number of cells injected), ascites tumors form in 100% of the vaccinated BALB / C mice. The amount of ascitic fluid accumulated in the abdominal cavity of mice after the administration of the IMB-4P7 hybridoma cells (an average of 20 animals) is 5 ml. The content of Mon-AT in 2 ml of ascitic fluid is 10-20 mg.

PRI me R 3. The use of Mon AT YMB-4R7 in enzyme immunoassay.

To determine the activity of

96-well plates sensitized with RNA polymerase of phage T7 (2.5 µg / ml protein, 100 µl of a thief per well). Then 100 µl of solution liquid is added to the lunctin in the necessary administration in 10 mM sodium phosphate containing 150 mm NaCl (PBS). To reduce non-specific interactions, 0.1% Tween-20 detergent is added to PBS and 0% bovine serum albumin is added to plates.

J5 is washed three times with PBS buffer, which burns 0.05% Tween-20 detergent (PBS-T).

After that, 100 µl of rabbit en

20 against immunoglobulins of the mouse with horseradish roxidase and incubated at 37 ° C. After a six-fold PBS-T kit, chromag substrate and 0.1% H2O2 are added to the wells.

25 citrate buffer (pH 4.0). Opt density of the substrate mixture is recorded at 405 nm using an eight-channel photometer and reports the results.

The use of the invention makes it possible to detect the DNA-dependent pH merase of bacteriophage T7.

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Claims (1)

Formula Invention Strain Hybrid Cultivated action of IMB-4R7 with antigenic animal detergent Mus musculus L The DNA-dependent RNA polymerase from bacteriophage T7 is mintated using the method of indirect solid phase analysis. As the solid phase is used ten 40157 96-well plates sensitized with RNA polymerase of phage T7 (2.5 µg / ml protein, 100 µl solution per well). Next, 100 µl of the solution of the ascic liquid in the required dilution in 10 mM sodium phosphate buffer containing 150 mm NaCl, pH 7.4 (PBS) is added to the wells of the plates. To reduce non-specific interactions in PBS, up to 0.1% Tween-20 detergent and 0.2% bovine serum albumin are added. Plates are incubated for 1 hour at 37 ° C and J5 three times washed with buffer SFR containing 0.05% detergent tween-20 (SFR-T). Thereafter, 100 µl of rabbit antibody conjugate is poured into the wells. 20 against mouse immunoglobulins with horseradish peroxidase and incubated for 1 h at 37 ° C. After six times washing with PBS-T, the chromogenic substrate and 0.1% H2O2 in 0.5 m are added to the wells. 25 citrate buffer (pH 4.0). The optical density of the substrate mixture in the wells is recorded at 405 nm using an eight-channel photometer and evaluate the results. The use of the invention allows for the detection of the bacteriophage T7 DNA-dependent RNA polymerase. thirty Invention Strain Hybrid Cultured BCKK (II) No. 35ID used to produce monoclonal antibodies to DNA-dependent RNA polymerase of bacteriophage T7.