SU1721092A1 - Strain of hydride cultured mammalian cells mus musculus l - a producer of monoclonal antibody to brain myelin glycoproteine, inhibiting activity of natural human killers - Google Patents

Abstract

This invention relates to hybridoma technology. Strain (III) of hybrid cells was obtained by fusing cells of Sp 2/0 mouse myelomas with spleen cells of a Balb / c mouse strain immunized with a brain glycoprotein associated with myelin. Hybrid cells produce class M immunoglobulins. Inoculation of cells of III cells in mice causes them to develop an ascites tumor. Specific antibodies are contained in the mouse ascitic fluid. The titer of ascitic fluid dilution in immunofermental analysis 10. The ability to produce antibodies is stably maintained for at least 16 passages. Monoclonal antibodies produced by W are capable of suppressing by 40% the natural killer activity of human peripheral blood mononuclear cells. The strain deposited under number VSKK (P) 453D.

Description

The invention relates to hybridoma technology and can be used in studying the effects of natural killer (EC) of human blood, as well as in medicine for diagnostic purposes and for assessing the immunological status of an organism.

Monoclonal antibodies (MonAt) are known that expose antigens on the surface membrane of EK cells. Among them are known MonAt, obtained using the glycoprotein (MAG) with a molecular weight as the antigen associated with myelin of the human brain. 110 kDa, on which epitopes recognized by MonAt Zeu-7 (or HNK-1), interacting with the human blood EK differentiation antigen HNK-1, are presented.

The purpose of the invention is to obtain a hybridoma strain producing MonAt to the myelin antigen of the brain, affecting the activity of natural killer cells (EC).

The hybridoma strain is obtained by hybridizing Sp 2/0 mouse myeloma cells with Balb / c mouse spleen cells. immunized with myelin-associated glycoprotein of the white matter of the human brain (MAG).

MAG is obtained from the white matter of the human brain and releases the drug from low molecular weight impurities according to known methods. From 5 g of the white matter of the human brain (cadaveric material), glyco- and lipoproteins are extracted by soft homogenization in a 20-fold volume of 0.32 M sucrose. Suspen VI

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The image is centrifuged in a density gradient of 0.32-0.85 M sucrose for 30 min at 75000 d. The precipitate formed at the interface of two media is washed with 40 times the volume of distilled water in 3 doses with centrifugation at 75,000-30000 d. Then the precipitate is suspended in 20-fold volume of 0.32 M sucrose and again subjected to gradient centrifugation under the same conditions as the first time. The resulting middle layer is dried. All procedures are performed at a temperature of 2-4 ° C.

1.5-2 g of the lyophilized protein are de-lipidized with 40 volumes of a mixture of chloroform, methanol (3: 1) for 1 hour with shaking. The suspension is centrifuged - Z.min at 75000d. The precipitate with ether (10 ml) is transferred to a flask, the ether is removed with nitrogen gas, and the solution is precipitated with 20 ml of 0.3 M L1-diiodsalicylate in 0.05 M cpm buffer pH 7.4. The solution is deproteinized with an equal volume of water saturated phenol. After deproteinization, phenol and LI-diiodosalicylate residues are removed by dialysis against distilled water, and then a physiological solution for a total of not less than 3 days (until the traces of lithium are removed according to the flame photometer). After dialysis, if the volume of the solution exceeds 8 ml, it is concentrated with ethylene glycol. The resulting dialysate is centrifuged for 1 hour at 10,000 g and then a fraction of 100-110 kDa is separated on an aqueous solution on Sephadex G200. This fraction is used as an antigen during immunization of mice and testing of monoclonal antibodies (MonAt).

Immunization of animals.

Balb / c mice aged 6–8 weeks are immunized according to the following pattern; The first injection is performed intraperitoneally - 50 µg of MAG with complete Froind-derived adjuvant per mouse. After 7 days, 25 µg of prepa. Rate were injected intraperitoneally with Freund's complete adjuvant per mouse. After another 4 days, for 3 days, 5 µg of the drug per mouse without adjuvant is administered intraperitoneally and intravenously. Two days after the last injection, the spleen cells are taken for fusion. Before fusion in the serum of immunized mice, the presence of antibodies to MAG is checked: a precipitation band was obtained in an agar precipitation reaction using TO μg of protein and twice the diluted serum. When tested by the enzyme immunoassay method, a positive reaction was obtained using 0.7 µg MAG per well and a 1: 1000 dilution of the blood serum.

Getting hybridoma. The splenocytes of the immunized mouse are fused with Sp 2/0 myeloma cells at a ratio of 5: 1 using polyethylene glycol (mol.m. 1500) in accordance with generally accepted methods. After fusion, the mixture of splenocytes with myeloma cells in a hybridoma medium is placed in 96-well panels coated with macrophages.

peritoneal exudate of B1b / s mice. On the following day, an equal volume of hybridoma medium with twice the amount of GAT is added to the wells. Hybridoma clones appeared on days 5-7 after merging in 50-70% of the wells. At this time, they start replacing the TAT environment. Testing of the presence in medium of MonAt to MAG was carried out by the immunofermental method 12–18 days after fusion. The identified producer cells are cloned onto the feeding layers of macrophages, cultured with Vitamin IH, or frozen with liquid nitrogen in a mixture of 90% fetal serum and 10% DMSO. Obtaining ascitic forms of hybridoma.

. Mice of the Ba1b / s line were administered intraperitoneally at 0; 5 ml of pristane. After 8-10 days, mice were cultured intraperitoneally with hybridoma cells in a solution of 0.14 M NaCI in 0.2 M phosphate buffer, pH

7.2 to 1x106 cells per mouse. After 7-8 days, ascites develops in mice, 2-7 ml of ascitic fluid is obtained from each mouse. Cells separated by gentle centrifugation (150 g, 7 min, sterile) are used for further hybridoma transplants, into ascites fluid as the source of MonAt.

 . Analysis of monoclonal antibody specificity.

The culture medium of the source and secondary hybridoma clones, as well as ascites fluid, is tested for the presence of MonAt to MAG using the ELISA method. When testing, 96 cell boards (Flow company) are used, in each well of which 0.5-0.6 µg of antigen (MAG) is added in 0.1 M carbonate buffer pH 9.5, and albumin is used as a blocking protein. RV solution pH 7.2.

Conjugate peroxidase with anti-mouse serum (Sigma) in dilutions of 1: 1000 on PBS with 0.5% albumin. The progress of the enzymatic reaction is judged by a change in the color of o-phenylenediamine (Sigma) in

diluted with 1 mg / ml 0.1 M citrate buffer pH 4.5. All flushing between separate

procedures carried out with a solution of PBS pH

7.2 in the presence of 0.05 MT of "wine-20 and water ..

The degree of developing color is evaluated in a multiscan spectrophotometer (Flow company) at a wavelength of 450 nm.

The interaction of MonAt with mononuclear surface antigens is determined by the method of the reaction of indirect immunofluorescence in a modification for test tubes.

For this purpose, 0.5 ml of the tested MonAt are added to test tubes of mononuclears isolated in a ficoll-urografin density gradient containing (3-4) x106 cells. The incubation time is 30 minutes at 37 ° C in an atmosphere of 5% C02. The cells are then washed 3 times in Hanks solution and G (AB) fragments obtained from luminescent anti-white mouse immunoglobulin serum are added (Research Institute of Microbiology named after A. Gamalei). The cells are resuspended and incubated for 30 minutes at 4 ° C. Then the cells are again washed 3 times in Hanks solution and placed on a cover glass. The preparations are dried and the cells are preserved with 50% glycerol on physiological saline. The registration of luminous cells on the preparations was carried out on a fluorescence microscope when viewing 300-500 cells.

When determining the cytotoxic activity of human EK cells, target cells use a cell culture of human editroblastosis K 562, labeled with H-uridine (35-40 Bq per TO cells). Lymphoid effector cells (mononuclear cells) are obtained from human donor blood in a ficoll-verografin density gradient. Mononuclear cells (7x10 cells in 0.5 ml), treated with MonAt in the complement fixation reaction, are incubated with target cells in a ratio of 1:25 in complete PPM medium for 16-18 h at 37 ° C in an atmosphere of 5% CO2. In the control variant together Monat, RPMI1640 nutrient medium is used. The remaining, after incubation, intact target cells are fixed on the filters and the radioactivity is counted in a toluene scintillor on a Vekmap counter. The cytotoxic activity of EK cells before and after their treatment with the ICA MonAt is judged by the change in the cytotoxic index determined by the formula

, radioactivity of an experimental sample,. „radioactivity in control x

It was shown that the obtained MonAt reduced by at least 40% the cytotoxic activity of the human blood mononuclear fraction in relation to the cells of the culture of human lymphoblasts C 562.

To determine the class of immunoglobulins of the test antibodies, the immunodiffusion method in agar is used according to the generally accepted method. MonAt with known and isotopes is used as control. Rabbit anti-mouse antibodies (RAM) are used as precipitating agent. The reaction is carried out in PEG containing 1% agar-agar with 2% PEG in 0.1 M PSR, pH 8.6. In the case of the isotope fusion — the studied and the known — the precipitation lines merge into one line, in this case IgM and IgM. In case of differences (IgM and IgG), neither occurs.

The physiological and cultural properties of the strain.

The strain is cultured on growth medium DMEM with the addition of 10% fetal serum, 1 mM of essential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, 5.10 M mercaptoethanol and 40 units / ml gentamicin at 37 ° C in an atmosphere of 5% C02. Suspension cultivation method. The population doubling time is 36 hours. The multiplicity of sowing is 1: 3.2 three times a week.

Cryopreservation is carried out in growth medium supplemented with 10% dimethyl sulfoxide at a concentration of (0.5-1). 10 cells in 1 ml. Cell viability of strain H 10/2 after thawing is 70%. Inoculation of cultured hybrid cells of strain H 10/2 to syngeneic Ba1b / s mice causes the formation of ascites tumor in them, ascites contain in liquid. MonAt to the differentiation antigen of HNK-1 EK cells. The capacity for MonAt production is maintained with several successive tumor transplants (at least 16). The titer At was determined in a dilution of ascites no less than 10. Cells remain viable when transferred to growth medium. When cultivated in vivo from one mouse, 2.5-7 ml of ascitic fluid (average 4 ml) is obtained.

The strain is deposited in the specialized collection of cell cultures of the Institute of Cytology, Academy of Sciences of the USSR - VSKK. The registration number of the deposit is 453D.

Claims (1)

Invention Formula The strain of hybrid cultured animal cells Mus musculus L No. VSKK (P) 453D is a producer of monoclonal antibodies to glycoprotein of brain myelin, which inhibits the activity of natural human killers.