RU2151184C1 - Strains of cultured murine hybridoma as producers of monoclonal antibodies raised to human erythtropoietin and antibodies producing by thereof - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: invention relates to hybridomas producing. Hybridomas are prepared by fusion of lymphocytes obtained from mice immunized with human erythropoietin with myeloma. Hybridomas are deposited at N VSKK/P/634D, VSKK/P/635D, VSKK/P/636D and they produce monoclonal antibodies raised to human erythropoietin. Molecular mass of monoclonal antibodies is 160 kDa and they belong to IgG1 with light chains of κ type. Invention ensures to obtain hybridoma panels producing antibodies to independent antigenic determinants of human natural erythropoietin. EFFECT: hybridoma strains indicated above, production of antibodies. 7 cl, 2 dwg, 1 tbl

Description

 The present invention relates to biotechnology, namely to hybrid cells and, in particular, to hybridomas. More specifically, the invention relates to hybridomas capable of producing monoclonal antibodies to human erythropoietin. These antibodies can be used for immunoaffinity purification of human erythropoietin, to isolate it from protein mixtures, or for its qualitative and quantitative analysis.

Known hybridoma strain of mice ATCC NHB8209, which is a producer of monoclonal antibodies to human erythropoietin [EP N 0116446, IPC ⁵ C 12 N 5/00, publ. 08/22/90]. The indicated strain was obtained by hyperimmunization of a Balb / C mouse with a cross-linked 20-membered synthetic oligopeptide synthesized by the authors. Strain HB 8209 produces monoclonal antibodies to an antigenic determinant in the structure of a synthetic 20-membered oligopeptide when it grows in a nutrient medium.

 A significant drawback of the known strain is the incomplete adequacy of the structure of the synthetic oligopeptide to the structure of the glycosylated human erythropoietin protein and, as a result, the inability to produce antibodies to erythropoietin with independently recognized antigenic determinants, which makes it impossible to use the obtained antibodies for the quantitative determination of erythropoietin. In addition, the fact described by the authors of the displacement of erythropoietin from the immune complex by a synthetic oligopeptide indicates a low affinity of the obtained antibodies for erythropoietin.

 The aim of the invention is to approximate the characteristics of the antibodies produced to the structure of natural human erythropoietin and to obtain a panel of hybridomas that produce antibodies to independent antigenic determinants of its molecule.

 This goal is achieved by cultivation of new strains of hybridomas of mice that received N BSC / P / 634D, N BSC / P / 635D and N BCC / P / 636D producing monoclonal antibodies to independent antigenic determinants of human erythropoietin upon registration. Strains obtained by the following technology.

 Human erythropoietin was immobilized on a HAHY type nitrocellulose membrane (Millipore, USA), the membrane was homogenized in dimethyl sulfoxide, nitrocellulose was salted with erythropoietin water, and the resulting suspension was introduced into Balb / c mice in the pads of the hind legs. After 30 days, the mice were boosted intraperitoneally with a solution of erythropoietin in an amount of 10 μg per 1 mouse. After 4 days, the mice were sacrificed and lymphocytes from the spleen and lymph nodes were used for subsequent hybridization. The fusion of lymphocytes from immune mice was carried out according to the standard procedure using polyethylene glycol. Myeloma partner Px63.Ag8.6.5.3 was used as a myeloma partner during fusion.

After fusion, cells were transferred to 96-cell microplates at a concentration of 10 ⁵ -10 ⁶ cells per well in a selective nutrient medium containing HAT. After 10 days, the microplates were microscopically monitored for the presence of growing cell colonies in the cells. Samples of culture fluid were taken from cells with growing colonies in order to identify colonies producing antibodies that react with human erythropoietin.

 Colonies producing antibodies against human erythropoietin were screened by enzyme-linked immunosorbent assay for erythropoietin immobilized on the surface of microplate cells for enzyme-linked immunosorbent assay (Costar, Holland, high-bond), according to the standard procedure (Engvall, E., and Pertmann J. Immunology, 109, 129, 1972). Cells from positive cells with growing colonies were subjected to cloning by transfer to new microplates at a concentration of 1 to 5 cells per cell. After 10-12 days, the presence of growing clones in the microplates was microscopically monitored, and culture fluid samples were taken from cells with growing colonies in order to identify clones producing antibodies to human erythropoietin. Positive clones were used for further cloning and propagated for culture deposition.

 Clones PCE / D7, PCE / D10 and PCE / F6 were selected from the initially positive colonies as a result of screening and cloning. These clones were characterized by a high growth rate, high stability in the production of antibodies that specifically react with human erythropoietin. Antibody production reaches 50 μg / ml when cultured under stationary conditions.

 The obtained strains of PCE / D7, PCE / D10 and PCE / F6 cultured by hybrid mice were deposited in the Specialized collection of transplantable somatic vertebrate cells of the All-Russian collection of cell cultures under the numbers VSCK / P / 634D, VSCK / P / 635D and VSCK / P / 636D, respectively.

Strains are characterized by the following features:
1. Morphological features
Cells of regular round shape, single or grouped into clusters.

2. Cultural traits
Type of growth - suspension
3. The principle of hybridization
The hybridoma was obtained by fusion of Px.63.Ag8.653 mouse myeloma with lymphocytes of Balb / C mice using polyethylene glycol.

4. Resistance to selective factors
Grows on selective HAT.

5. Cryopreservation
Cryopreservation medium - 90% fetal serum and 10% dimethyl sulfoxide. Storage at -196 ^o C in liquid nitrogen.

6. Control of species identity
The hybridoma is capable of cultivation in the peritoneal cavity of syngeneic mice without the use of immunosuppression and produces mouse immunoglobulins of the type Hγ1 L _k .

7. Contamination
Hybridoma cells are free of mycoplasma.

Cumulative cultivation of the obtained hybridomas in a serum-free synthetic culture medium was carried out. Culture liquids in an amount of 1 liter for each clone were concentrated by ultrafiltration on a hollow fiber module with a fiber permeability of 30 kD to 100 ml and diafiltered against 20 volumes of 0.02 M Tris buffer pH 7.5. The obtained concentrates were frontally applied to a K26 / 30 column containing 100 ml of Q-Sepharose FF anion exchanger (Pharmacy, Sweden), equilibrated with the same buffer. The antibodies were eluted by a linear gradient of sodium chloride in the same buffer from 0 to 0.5 M. Collected major peaks containing monoclonal antibodies were applied to a K26 / 100 column filled with Sephacryl S-200 gel (Pharmacy, Sweden) and eluted 0 , 02 M phosphate buffer pH 7.2, containing 0.15 M sodium chloride. As a result of purification, 12 mg of monoclonal antibodies in an almost homogeneous state were obtained. Monoclonal antibodies were characterized by the following parameters:
Using the Calbiochem enzyme immunoassay kit, isotypes (γ1) of the heavy and light (k) immunoglobulin chains were established.

 Enzyme immunoassay has shown the ability to bind human erythropoietin from a solution.

 Using enzyme-linked immunosorbent and immunofluorescence analysis, it was found that monoclonal antibodies produced by the claimed strains do not bind components of human and bovine serum and human spleen cell proteins. It was also found that antibodies do not bind the cell proteins of the recipient strain of CHO, used to obtain a cell line producing erythropoietin.

Using electrophoresis, the resulting antibodies were characterized by molecular weights (160 kD) and purity. The results are presented in the table and figure 1
In FIG. 2 presents a spectrum of isoelectric focusing of monoclonal antibodies produced by the claimed strains. As follows from the figure, antibodies are characterized by isoforms located in the pH range from 5.3 to 6.0.

Claims (6)

 1. The strain of cultured hybridomas of mice N VSCK / P / 634D - producer of monoclonal antibodies PCE / D7, specifically interacting with human erythropoietin.  2. Monoclonal antibody PCE / D7, specifically interacting with human erythropoietin, characterized by the following features: produced by hybridoma of mice N VSCK / P / 634D according to claim 1; has a spectrum of isoelectric focusing bands in the range of P1 5.7 - 6.0; belongs to the class IgG1.  3. The strain of cultured hybridoma of mice N VSCK / P / 635D - producer of monoclonal antibodies PCE / D10, specifically interacting with human erythropoietin.  4. Monoclonal antibody PCE / D10, specifically interacting with human erythropoietin, characterized by the following features: produced by hybridoma of mice N VSCK / P / 635D according to claim 3; has a spectrum of isoelectric focusing bands in the range of P1 5.3 - 5.7; belongs to the class IgG1.  5. The strain of cultured hybridoma of mice N VSCK / P / 636D is a producer of PCE / F6 monoclonal antibodies that specifically interact with human erythropoietin.  6. Monoclonal antibody PCE / F6, specifically interacting with human erythropoietin, characterized by the following features: produced by hybridoma of mice N VSCK / P / 636D according to claim 5; has a spectrum of isoelectric focusing bands in the range of P1 5.3 - 5.7; belongs to the class IgG1.

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