RU2008350C1 - HYBRID STRAIN OF ANIMAL MUS MUSCULUS CELLS FOR PRODUCING MONOCLONAL ANTIBODIES AGAINST HUMAN IgG - Google Patents

Abstract

FIELD: biochemical technology. SUBSTANCE: hybrid strain of animal Mus musculus cells which is producer of anti-human IgG monoclonal antibodies is produced by hybridization of IgG antigen-pulsed spleen macrophages of Balb 1c mice with myelocytes Sp 2/0 Ag. 14. Monoclonal antibodies is anti-human IgG 2a antibodies having the capacity of site-specific binding of human IgG-type molecules except their Fab-Fc fragments. The titers of such monoclonal antibodies are of 5-10 μg/ml in liquid culture medium and about 5-10 mg/ml in ascite liquid. EFFECT: increased antibody-producing capacity. 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 (MCA), interacting with human IgG1, 2, 3, 4, as well as their Fab-, Fc- [4] and only Fc-fragments [1]. A distinctive property of the proposed strain is the production of antibodies that interact with whole molecules of IgG1, IgG2, IgG3, IgG4 of the person in the hinge region and do not react with Fab-, Fc-fragments of IgG.

 For hybridoma fusion earlier [4, 1], NS1 myeloma cells were used, which, unlike the Sp 2/0 Ag14 line used in the invention, 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 is known that each newly obtained hybridoma has strictly individual parameters. MCAs produced by different hybridomas differ in their primary structure, specificity for various antigenic determinants, affinity, ability to bind complement, cytotoxicity, hemagglutinating and many other properties. The use of several MCAs for immunoglobulins in the complex expands the possibilities of their use in biochemical, immunological and clinical studies. Therefore, obtaining MCAs for different antigenic determinants of human immunoglobulins, which are the central link of humoral immunity, allows their use in various variants of immunological methods.

 The technical result of the invention is to obtain a hybrid strain of cultured cells of Mus musculus L. producing MCA to an antigenic determinant located in the hinge region of 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 gastric adenocarcinoma by ammonium sulfate reprecipitation followed by ion exchange chromatography on DEAE cellulose) in 0.005 M phosphate buffer solution with 0.15 M NaCl, pH 7.4 PBS) with Freund's complete adjuvant. Immunization is repeated after 4 weeks without adjuvant. 4 weeks after the second immunization, mice are boosted by the intravenous administration of 100 μg IgG in PBS. After 3 days in animals, the titer of antibodies to human IgG is determined by enzyme-linked immunosorbent assay. A mouse with a better immune response is used to produce a hybridoma. For this, 10 ⁸ spleen cells of the immune mouse hybridize with 4 x 10 ⁷ Sp 2/0 Ag14 myeloma cells 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 twice 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 against human IgG. The most productive clone is introduced into mass culture and designated H11G5.

 Strain H11G5 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 inoculated into the peritoneal cavity of BALB / 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 of 5 x 10 ⁵ cells per 1 ml. Multiplicity of sieving 1: 5, subculture time 3-4 days. The culture medium is RPM1-1640 with the addition of 10% fetal bovine serum, 2 mM / L glutamine and pyruvate, 100 units / ml penicillin and streptomycin. In the first 3 weeks after hybridization, 10 ^-4 M hypoxanthine, 4 x 10 ^-7 M aminopterin and 1.6 x 10 ^-5 M thymidine are added to the medium, since Sp 2/0 Ag14 myeloma cells are defective in the hypoxanthine guanine phosphoribosyltransferase enzyme. Cells are grown in an atmosphere of 5% CO ₂ at 37 ^about C.

 Cultivation in the body of the animal. MCA is produced by culturing in vivo cells on BALB / c mice (pretreated 0.5 ml with tetramethylpentadecane intraperitoneally) in an amount of 1-2 million cells per mouse. Tumors form 10-14 days after vaccination.

 The characteristic of a useful product. MAB produced by the H11G5 strain belong to the IgG2a subclass, specifically interact with the human IgG in the hinge region. The affiliation to the IgG subclass is determined using precipitating sera against IgG from mice of different subclasses (Sigma, USA) by Ouchterloni radial immunodiffusion. The interaction of antibodies with human IgG is determined by enzyme-linked immunosorbent assay.

 The productivity of the strain, the stability of antibody production. The concentration of MCA after 3-4 days of cultivation "in vitro" 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 productivity is maintained for 25 passages in culture and 15 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. Freezing mode: 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. Hybridoma cells are placed in a 25 cm ² plastic bottle of 5 x 10 ⁵ cells in 5 ml of RPM1-1640 medium supplemented with 10% fetal bovine serum, 2 mM / L glutamine and pyruvate, 100 units / 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 MAB to human IgG is used in an enzyme-linked immunosorbent assay. Various immunoglobulins isolated from the blood serum of healthy people and patients with myeloma (using the method of reprecipitation with ammonium sulfate followed by ion-exchange chromatography on DEAE-cellulose) are used as antigens: IgG don, IgG1 Kuz, IgG1 Mys, IgG1 Rosh, IgGG2 Bob, IgG2 Bob, IgG2 Bob lgG2 b / n, lgG3 Dal, lgG4 Zhel, lgG4 Ber, lgA, as well as lgM (isolated from the blood serum of a patient with Waldenstrom's disease by reprecipitation with bidistilled water followed by gel filtration) and pipain Fab, Fc fragments of human lgG. 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 hour 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 1 hour, the wells emptied, the wells were washed with cold water and PBST, was added 50 .mu.l of conjugate-rabbit lgG antibodies to mouse peroxidase diluted 1: 500 in PBST solution and incubated at ³⁷ ° C for 1 hour After thorough washing. a 0.4 mM solution of the substrate, 2.2 ^I- azino-di- [3-ethyl-benzothiazoline sulfonate (6)] (ABTS) in 0.05 M Na-citrate buffer, pH 4.0 with addition of hydrogen peroxide was added to the wells to a final concentration of 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 H11G5 hybridoma produce MCAs that specifically interact with the human IgG antigenic determinant located in the hinge region. (56) 1. Lowe J., Bird P., Hardie P. et al. // J. Immunol - 1982, v. 47 (r). - p. 221-396.

 2. Milstein C., Aletugbo K., Cowan N. J. et al. // Cold Spring Harbor Symp. Quabt Biol. -1976. -v. 41, p. 793.

 3. Kohler O., Howe S. C., Milstein C. // Eur. J. 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. VSCK (P) N 491 D used to obtain monoclonal antibodies to human IgG.

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