RU2018531C1 - Strain of hybrid cultured mammalian cells mus musculus l used for preparing of monoclonal antibodies to glycoprotein antigen 70 kda of cattle peripheral blood mononuclears - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves the preparing of strain of hybrid cultured mammalian cells Mus musculus L. producing monoclonal antibodies to glycoprotein antigen of cattle peripheral blood mononuclears. Molecular mass of antigen is 70 kDa. Strain is prepared by hybridization of murine splenocytes (strain BALB/c) with myeloma cells NSO. Monoclonal antibodies are IgG-class. Stable production of antibodies is retained in vitro for 10 passages. Monoclonal antibodies can be used for detection and study of membrane-bound antigens of cattle peripheral blood mononuclears. EFFECT: preparing of strain indicated above.

Description

 The invention relates to biotechnology and can be used to develop immunodiagnostics and experimental studies.

 Known strains of hybrid cells producing monoclonal antibodies (MABs) to lymphocytes in cattle [1].

 Both strains were obtained by immunizing mice with mononuclear cells from the peripheral blood of a cow with leukemia, while the proposed strain is obtained by immunizing mice with mononuclear cells isolated from the peripheral blood of a healthy cow. In addition, the antigenic specificity of known antibodies has not been established.

 The technical result of the invention is to obtain a strain of hybrid cultured animal cells of animals Mus musculus L., producing monoclonal antibodies to glycoprotein 70 kDa of mononuclear peripheral blood of cattle (cattle).

 The strain is obtained as follows.

Murine NSO mouse myeloma cells hybridize to spleen cells of Balb / c mice immunized with cattle peripheral blood mononuclear cells. Use the following immunization schedule: 1 x 10 ⁷ mononuclear cells / mouse intravenously (iv) 3 times with an interval of 14 days. After the 3rd immunization, the titer of antibodies in the blood serum of mice is determined and boosting is carried out in the same way and with the same dose of antigen. After 4 days, the mouse is sacrificed, the spleen is removed, and myeloma cells and splenocytes of the immune mouse are hybridized in the presence of polyethylene glycol (M = 4000). The selection of hybrid cells is carried out using a special medium containing hypoxanthine, aminopterin, thimedin (GAT)
As a result of testing the obtained hybrid clones on the basis of the production of antibodies to glycoproteins from peripheral blood mononuclear cells of cattle and their subsequent reclamation, a strain is selected that produces MCAs of the required specificity.

 The resulting strain is characterized by the following features.

 Cultural properties.

 The medium for the cultivation of the strain is Dulbecco's Eagle medium (DMEM) supplemented with 15% fetal calf serum, 4 mM L-glutamine, 80 U / ml gentamicin.

Cultivation was carried out at ³⁷ ° C in an incubator with 5% carbon dioxide, in an atmosphere of 95% humidity and in sealed vials in an incubator without _CO2 supply.

Cultural vials are used to grow the strain. In a bottle of 25 square meters. cm ² in 5 ml of medium seeded 1 x 10 ⁶ cells. Passage 1 time in 2-3 days with the transfer of 1/3 cells without counting.

For growing ascites, Balb / c mice are used. 2 weeks before the injection of the strain, the mice are injected intraperitoneally (ip) with 0.5 ml of liquid paraffin. The cells of the strain are injected ip (1-2 x 10 ⁶ cells per mouse). Ascites are collected after 8-12 days, 3-7 ml from each mouse.

 The characteristic of a useful product.

 The strain produce mAbs related to IgG. They bind superficial cattle peripheral blood mononuclear glycoprotein. The molar mass of antigen is 70 kDa. MCA are formed in vitro on the 7th day of cultivation. Stable antibody production persists for 10 passages.

 Contamination control.

 Bacteria, fungi and mycoplasmas were not detected during prolonged observation and culture on culture media.

The method of cryopreservation. Strain cells were resuspended in DMEM containing 50% fetal serum, 10% DMSO, and a concentration of 3 x 10 ⁶ cells in 1 ml are placed into 1 ml vials at 4 ° ^C. Then the ampoule was frozen by the application: lowering the temperature by 1 ^{° C} per minute to -40 ^{° C} and 10 ^{° C} per min to -196 ° ^C. Defrosting cells: 1 min at 37 ^C. the cells were diluted into 10 mL complete media, pelleted by centrifugation, resuspended in 5 ml of the same medium with 10 % fetal calf serum and transferred to a culture bottle.

 Cell viability for trypan blue inclusion is 68%.

PRI me R 1. Blood from the jugular vein of cattle taken in tubes with an anticoagulant (25-50 IU / ml heparin), diluted 2 times with buffered saline (EGF) and layered on the gradient of verographinficoll (d = 1,077). Centrifuged for 45 minutes at 2000 rpm. Mononuclear cells are removed from the gradient, washed in PBS. Single red blood cells (if any) are removed using 0.83% ammonium chloride in a ratio of 1: 9. The mononuclear cell pellet was washed in PBS (2000 rpm, 7 min). Glycoproteins are isolated by lysing mononuclear cells with a detergent, followed by purification by affinity chromatography. To this end 1 x ¹⁰ August mononuclear cells were lysed with 0.5% NP-40 detergent in 10 mM Tris HCl / pH = 8,0 (for 30 minutes at 4 ° ^C. The lysate was centrifuged at 12,000 rpm / 30 min min, then determine the protein content in the supernatant on a spectrophotometer at λ = 280 nm.

 The volume of a solution containing 30 mg of protein is applied to a 5 ml column with concanavalin A - Sepharose. The column is thoroughly washed with 0.1 M phosphate buffer (pH = 7.4) with 0.2% sodium deoxycholate, then the glycoproteins are eluted with 0.1 M methylmannopyranoside, the protein from the eluate is precipitated with ethanol, dried and dissolved in 0.1 M phosphate buffer in concentration of 0.5 mg / ml.

 Affinity purified glycoproteins from peripheral blood of cattle are used at a concentration of 5 μg / ml.

To obtain MCA, the cells of the strain are inoculated with syngenic Balb / c mice. Each of 10 mice is pre-injected intraperitoneally (ip) with 0.5 ml of liquid paraffin. After 14 days, the animals were injected ip in 1-2 x 10 ⁶ cells / mouse.

 After 8-12 days, ascites fluid is removed from the abdominal cavity of mice, 3-7 ml from each mouse. Cells are separated by centrifugation (2000 rpm, 10 min), and the supernatant is used as a source of MCA.

 After electrophoresis of glycoproteins from peripheral blood mononuclear cells of cattle, the protein is transferred to nitrocellulose at a current of 500 mA in an electroblotting chamber in an electrode buffer containing 25 mM Tris, 192 mM glycine, 20% methanol (pH = 8.3) for 3 hours. I block free spaces on nitrocellulose for 2 hours with a solution containing 0.5 M NaCl, 10 mM Tris HCl (pH = 7.5) (TPS) and 1% gelatin. After electrotransfer, immunological detection of proteins on nitrocellulose is carried out. Nitrocellulose and ascitic fluid containing 1: 1000 diluted MCA are transferred to TBS with 0.2% Tween-20 for 4 hours. The bound MCAs are visualized using rabbit antibodies to mouse immunoglobulins conjugated to horseradish peroxidase. For color detection of peroxidase reactions using diaminobenzidine. The research results showed that the studied MCPs bind to the material from the peripheral blood mononuclear cells of cattle in the zone of 70 kDa.

 PRI me R 2. The surface localization of antigens specific for the obtained antibodies is determined using the complement dependent lymphocytotoxic test (CCT). It is as follows.

In the wells of the Terasaki microcameras, 1 μl of the studied antibodies is added under a layer of petroleum jelly, then 1 μl of the suspension of peripheral blood lymphocytes of cattle (2 x 10 ⁶ cells / ml) .. The cells are incubated for 0.5 h at room temperature. Then, 2 μl of rabbit complement was added to the wells. The chambers are incubated for 1 h at room temperature. Then, 2 μl of 0.25% trypan blue are added to the wells. The reaction is taken into account after 4-5 minutes under a microscope. The intensity of the reaction is expressed in crosses on a four-point system.

 1 + (1-25%) dead cells, 2 + (26-50%) dead cells, 3 + (51-75%) dead cells, 4 + (76-100%) dead cells.

 The studied Mon MCAs weakly react in CTT with peripheral blood lymphocytes of cattle (1+). They bind 5-25% of cattle blood lymphocytes. Thus, the proposed MCP can be used to identify and study membrane-bound antigens of peripheral blood mononuclear cells of cattle.

Claims (1)

 The strain of hybrid cultured animal cells Mus musculus L. VSCC (P) N 555D used to obtain monoclonal antibodies to the glycoprotein antigen of 70 kDa of cattle peripheral blood mononuclear cells.