RU2010856C1 - Strain of hybrid cultured mammalian cells mus musculus l, used for preparing of precipitating monoclonal antibodies to human igg - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves preparing of strain of hybrid cultured mammalian cells Mus musculus L. producing precipitating monoclonal antibodies to human IgG. Strain is prepared by hybridization of murine splenocytes (line Bal b/c) with myeloma cells Sp2/OAg14. Monoclonal antibodies belong to subclass IgG1, they interact with human IgG specifically and show precipitating properties. Concentration of monoclonal antibodies in cultural fluid is 5-10 micro g/ml and in ascitic fluid is 5 mg/ml. Production of antibodies continues for 30 passages in culture and for 25 passages in animals. EFFECT: preparing of strain indicated above. 1 tbl

Description

 The invention relates to biotechnology and can be used for structural and functional studies of human immunoglobulins G IgG, the development of immunodiagnostic drugs in biology and medicine.

 Known strains producing monoclonal antibodies (MCAs) to human IgG [1, 4]. A distinctive feature of the proposed MCA is the ability to precipitate human IgG. This property allows the use of MCA of this strain in various immunodiffusion reactions for the diagnosis of myeloma, autoimmune and other diseases, accompanied by a change in the quantitative ratios of protein fractions.

For hydridomic fusion [1, 4], NS ₁ myeloma cells were used, which, unlike Sp 2/0 Ag 14, synthesize immunoglobulin light chains that can integrate into antibody molecules [2], which can lead to inactivation of some antibodies [3 ] and, accordingly, reduce the effectiveness of the application for enzyme immunoassay.

 It should also be noted that each newly obtained hybridoma is unique. MCAs produced by different hybridomas differ in their primary structure, specificity for various antigenic determinants, affinity, ability to bind complement, cytotoxicity, hemagglutinating and other properties. Therefore, obtaining as much as possible of MCA for human IgG is important from a scientific and practical point of view.

 The technical result of the invention is to obtain a strain of hybrid cultured animal cells of animals Mus musculus L., producing precipitating MCA for human IgG.

 The strain is obtained as follows.

BALB / c mice were immunized by intraperitoneal administration of 100 μg IgG (isolated from serum of a patient with myeloma disease by ammonium sulfate reprecipitation followed by ion exchange chromatography on DEAE cellulose) in a 0.005 M phosphate buffer solution with 0.15 M NaCl pH 7.4 (РS ) with Freund's complete adjuvant. Immunization is repeated after 4 weeks, but the antigen is administered without adjuvant. After 4 weeks, mice are boosted by intravenous administration of 100 μg IgG in PBS. After 3 days in animals, the titer of antibodies to human IgG is determined by enzyme immunoassay. A mouse with a better immune response is used to produce a hybrid home. For this, 10 ⁸ cells of Sp 2 / p Ag14 myeloma in the presence of a 50% solution of polyethylene glycol per mol. m. 4000 (Merck, Germany). After hybridization, cells are seeded in 96-well plates at 10 ⁵ cells per well. The peritoneal macrophages of the mouse are used as the feeding layer, which are seeded with 10 ⁴ cells per well one day before hybridization. The hybridoma is cloned 2 times by limiting dilution (based on 32 cells per 96-well plate), plating cells on a layer of peritoneal macrophages. After the second cloning, more than 90% of the obtained subclones produce antibodies to human IgG. The most productive clone is introduced into mass culture and designated 2FIIEII.

 Strain 2FIIEII is characterized by the following features.

 Morphological signs. The type of strain growth is suspension, cells of a rounded shape, arranged in clusters. The hybridoma causes the formation of mixed ascites and solid tumors when vaccinated in the peritoneal cavity of BAL B / c mice at a dose of 1 million cells per mouse.

Cultural signs. The hybridoma is cultivated in plastic bottles of 50-250 ml. Sowing dose - 5 ^. 10 ⁵ cells per 1 ml. Multiplicity of sieving 1: 5, subculture time 3-4 days. Cultivation medium RPMI-1640 with the addition of 10% fetal bovine serum, 2 mm glutamine, 2 mm pyruvate, 100 units / ml penicillin and streptomycin. In the first 3 weeks after hybridization, 10 ^-4 M hypoxanthine, 4, is added to the medium ^. 10 ^-7 M aminopterin and 1.6 ^. 10 ^-5 M thymidine, since Sp 2/0 Ag14 myeloma cells are defective in the enzyme hypoxanthine-guanine phosphoribosyltransferase. Cells are grown in an atmosphere of 5% CO ₂ at 37 ^about C.

Cultivation in the body of the animal. To grow hybridomas in ascites form, cells (10 ⁷ ) are injected into the abdominal cavity of BAL B / c mice, which previously (for 7-10 days) injected pristane intraperitoneally. The formation of ascites is 10-14 days.

 The characteristic of a useful product. MAB produced by strain 2FIIEII belong to the IgGI subclass, specifically bind human IgG and, in a mixture with MAB hybrid 2HIIF9 or 3H2D2, precipitate this antigen. Membership in the IgG subclass is determined using precipitating sera against mouse IgG of different subclasses (Sigma, USA) by Ouchterloni radial immunodiffusion. The ability to precipitate with human IgG is determined by enzyme immunoassay.

 The productivity of the strain, the stability of antibody production. The concentration of MCA after 3-4 days of in vitro cultivation is 5-10 μg / ml, in ascites fluid 5 mg / ml. Productivity control is carried out using enzyme-linked immunosorbent assay, titer 1/5000. Antibody production is maintained for 30 passages in culture and 25 passages in animals.

 Contamination control. Bacteria and fungi were not found in the culture during long-term observation and culture on culture media. Mycoplasma infection was not detected when stained with DNA dyes.

The method of cryopreservation. For long-term storage, the cells of the strain are frozen in fetal bovine serum with the addition of 10% dimethyl sulfoxide. Regis freezing: hours at ^-70 ° C, then cells were transferred to ampoules in liquid nitrogen. Cells were thawed by transferring an ampoule from liquid nitrogen in a water bath at 37 ^C. Cell viability after thawing is 70-80% by trypan blue staining.

PRI me R 1. Hybridoma cells are placed in a plastic bottle with an area of 25 cm ² to 5 ^. 10 ⁵ cells in 5 ml of RPMI-1640 medium supplemented with 10% fetal serum, 2 mM glutamine and pyruvate, 100 u / ml penicillin and streptomycin. Cells were cultured for 3-4 days at ³⁷ ° C in an atmosphere containing 5% CO _2. The culture medium is harvested, centrifuged for 10 minutes at 800 rpm. The supernatant containing the MCA for human IgG is used in an enzyme-linked immunosorbent assay. Various immunoglobulins isolated from the blood serum of healthy people and patients with myeloma disease by ammonium sulfate reprecipitation followed by ion exchange chromatography on DEAE cellulose are used as antigens: IgG don, IgGI Kuz, IgGI Mis, IgGI Goal, IgG2 Pisces, IgG2 Bob, IgG2 Bob, IgG2 Bob, IgG2 Bob, IgG2 Bob, IgG2 Bob b / n, IgG3 Dal, IgG4 Zhel, IgG4 Ber, IgA, as well as IgM isolated from the blood serum of a patient with Waldenstrom's disease by reprecipitation with bidistilled water followed by gel filtration, and papain Fab-, Fc-fragments of human IgG. Antigens are diluted to a concentration of 10 μg / ml in 0.05 M Na-carbonate buffer, pH 9.2 and add 50 μl to a well of 96-well plates. Incubate for 1 hour at ³⁷ ° C, then decanted and make 100 l per well 1% solution of bovine serum albumin (BSA) for 1 h at 37 ^C. After incubation, BSA solution was decanted, the plate was washed with cold water and PBS with 0.05% Tween (PBST), the supernatant of the culture medium is made with 50 .mu.l per well and incubated at 37 ° ^C. After one hour the wells was emptied and the wells washed with cold water and RVST, was added 50 .mu.l of conjugate rabbit antibodies to mouse IgG peroxidase diluted 1: 500 in PBST solution and incubated at ³⁷ ° C for 1 hour After thorough rinsing. In the wells, a 0.4 mM solution of the substrate, 2,2-azino-di-3-ethylenebenzothiazoline sulfonate (6)] (ABTS) in 0.05 M Na-citrate buffer, pH 4.0 with the addition of hydrogen peroxide to a final concentration is added to the wells 0.01% and incubated on a shaker at room temperature for 30 minutes. Then evaluate the result on a Multiskan spectrophotometer (Flow, UK) at a wavelength of 405 nm.

 The data obtained, expressed as a percentage of the maximum extinction (1.0-100%), are presented in the table.

 The data presented in the table indicate that the hybridoma 2FIIEII produces MCAs that specifically interact with human IgG.

 PRI me R 2. A 3% agar solution in PBS was poured onto a glass plate. Punches form holes at a distance of 0.6 cm from each other. The ascitic fluid of strain 2FIIEII is mixed with the ascitic fluid of strain 2HIIF9 or 3H2D2 in a ratio of 1: 1 and introduced into the central wells. IgG, IgA, IgM, BSA are added to the lateral wells. After 24 hours, upon interaction of IgG with a mixture of 2FIIEII with 2HIIF9 and with a mixture of 2FIIEII with 3H2D2, precipitation bands are formed that do not form in the case of interaction of antibodies with IgA, IgM, BSA.

 Thus, the proposed hybrid strain produces precipitating MCAs that specifically interact with human IgG. The resulting antibodies can be effectively used in biochemical and clinical studies. (56) 1. Lowe I., Bird P., Hardie D. et al. I. Immunol. - 1982. - v. 47 (2). - p. 221-396.

 2. Milstein C., Adetigbo K., Cowan N. I. et al. Cold Spring Harbor Symp. Quant Biol. - 1976. - v. 41. p. - p. 793.

 3. Kohler O., Howe S. C., Milstein C. Eur. I. Immunol. - 1976. - v. 6, - p. 292.

 4. European patent N 0163141, CL C 12 P 21/00, 1985.

Claims (1)

 The strain of hybrid cultured animal cells of Mus musculus L. HSCC (P) N 566 D used to obtain precipitating monoclonal antibodies to human Ig G.

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