SU1685997A1 - Strain of hybridous cultured mammalian cells mus musculus l - a producer of monoclonal antibodies to human thyrotropin - Google Patents

Abstract

The invention relates to hybridoma technology and may be. used for the quantitative determination of human thyroid-stimulating hormone (TSH) by immunoassays. The purpose of the invention is to obtain a strain of hybridoma cells of a mouse, producing monat, specific for TSH and not giving cross-reactions with other pituitary hormones: FSH, LH, STH and hCG. MonAT was selected for specificity by the method of solid phase immunoassay. The strain is cultivated in RPMI 1640 medium supplemented with 10% bovine fetal serum, 2 tM L-glutamine and 5-10 M 2-mercaptoethanol. The strain is grafted into the abdominal cavity of syngeneic mice in the form of ascitic tumors. MonAT belong to the IgGI subclass. Purified monat used as the 1st layer on the solid phase and ELISA on TSH. As 2 antibodies (above), anti-TSH antiserum rabbit antisera is used. Such a test system allows the determination of TSH in the range of 0.4-50 mIU / ml. The strain deposited under number VSKK (P) 366D. (A C

Description

This invention relates to hybridoma technology and can be used to quantify human thyroid stimulating hormone (TSH) by immunoassays.

The purpose of the invention is to obtain a monAT producing strain that is not inferior in prototype specificity.

Strain T-7, number VSKK (P) 366 (D) was obtained as follows.

Hybridoma is obtained by fusing splenocytes of mice immunized with human TSH with myeloma cells.

line X63 Ad8653 mouse. For immunization, BAIB / c females are used. Immunization is carried out by triple introduction into the abdominal cavity with an interval of 13 days of a nitrocellulose filter with applied TSH in an amount of 0.5 µg. The immunization process is monitored by taking blood and checking for the presence of antibodies with TSH using ELISA. Hybridization of the cells was carried out with PEG (m, v. 4000). The ratio of splenocytes and myeloma cells is 5: 1. After fusion, the cells are transferred to the GAT selective medium (hypoxanthine-amyoneh

ABOUT

nopterin thymidine) and cultured until clones appear in complete RPMI 1640 medium containing 5% fetal bovine serum and 5% reindeer serum, 2 mM L-glutamine, 5x10 M 2-mercaptoethanol, 10 4 M hypoxanthine, 4 x M aminopterine and 1.6x10 M thymidine.

The specificity of hybridoma antibodies is determined by the method of enzyme-linked immunosorbent assay (ELISA).

For ELISA, 96-well polyvinyl chloride (PVC) microtiter plates are coated with TSH hormone solutions, follicle-stimulating (FSH), lutenizing (LH). somatotropic (STG) and choriogonadotropic (CG) in carbonate-bicarbonate buffer 0.05 M, pH 9.6, 50 μl per well and incubated overnight at 4 ° C or at 37 ° C for 1 h. Then the wells of the plates are blocked with a 0.5% solution of gelatin in PBS at 37 ° C for 40 minutes, followed by washing with a PBS solution of 0.05% Tween-20. Hybridoma supernatants are introduced into the wells of hormone-coated plates. Mouse antiserum (dilution 1: 10,000) was used as a positive control, and RZ-X63 myeloma supernatant was used as a negative control. Ad8653. After incubation for 1 hour at 37 ° C, the plates are washed three times with PBS / Tween-20 and high-specific rabbit antibodies against mouse immunoglobulins conjugated with horseradish peroxidase are added and the plates are incubated for another 40 minutes at 37 ° C. After washing the PBS / Tween-20 plates three times, a 0.04% solution of o-phenylenediamine in 0.2 M phosphate-citrate buffer pH 5.0 was added to the wells with the addition of 0.012% hydrogen peroxide. After the occurrence of color in the wells, the reaction is stopped with a solution of 2n. sulfuric acid. Spectrophotometric determination of the reaction is carried out at 492 nm. From the results it follows that monat T-7 is specific only to thyroid-stimulating hormone. Cultures of hybridomas, in the supernatants of which antibodies against TSH were detected, which do not react with FSH, human HCG, STH and human LH, are cloned by limiting dilution of 0.3 cells per well in RPMi 1640 with 10% fetal calf serum, 2 mM glutamine , 2-mercaptoethanol in the presence of hypoxanthine (10 h M) and thymidine (1,).

The strain is characterized by the following properties.

Cultural properties. The cells are cultured in the urine at a concentration of 5x10 10 cells / ml, the subculturing time is about 4V h

Cryoconservation Cryopreservation of T-7 hybridoma is carried out in ETS containing 15% DMCO. The first 3 sug ampoules with cells are stored at -70 ° C, then transferred to liquid nitrogen. The viability of hybridomas is assessed by counting the cells when stained with their 0.5% solution.

0 trypan blue. Viability of about 80%.

Characterization of monoclonal antibodies.

The heavy and light chain isotypes were determined by direct enzyme immunoassay using the isotype-specific antibody conjugates as developing agents. MonAT series T-1 have light chains of type K, isotype heavy

0 chains - gamma 1. The strain is able to grow in the body of syngeneic mice in the form of an ascitic tumor.

For the production of ascites, BALB / c mice from females of 12–14 weeks of age are primed with 0.5 ml intraperitoneal injection 10 days before inoculating them with hybridoma cells. Hybridoma cells washed with RPMi 1640 medium are injected intraperitoneally with 2x10 cells per mouse.

Ascites formation 14-20 days. monat concentration 2-10 mg / ml.

The isolation of monoclonal antibodies is carried out from ascitic fluid using ammonium sulfate fractionation and

5 subsequent chromatography on DEAE cellulose. The homogeneity of the isolated monoclonal antibodies was checked by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. To prove the monoclonal nature of antibodies, an analytical iso-electrofocusing method in a thin layer of a pyacrylamide gel is used. Determined that

5, all antibodies of the T-7 series are products of individual clones of cells and are isolated on electrophoregrams as a set of closely spaced bands with isoelectric points in the ranges

0 pH 6.7-8.0.

The resulting strain of hybrid cultured T-7 cells is stored in the Specialized Collection of Transplanted Somatic Vertebrate Cells of the All-Union Collection of Cell Cultures under the number of HSC (S) 366D.

Example. Determination of human TSH using monat T-7. Monoclonal antibodies T-7 adsorb on microtitre PLEAT plates in distilled water

(pH 4.7) with a concentration of 40 μg / ml. Then, 100 ml of PBS containing 0.5% gelatin are added to the wells and incubated for 1 hour at 37 ° C. Then, 50 ml each of standards containing a known amount of TSH (100. 50 and 25 mIU / ml) are added to the wells. After incubation for 1 hour, 50 ml of rabbit polyclonal antibodies specific for human TSH are added to the wells in a 0.2% PBS / gelatin solution at a concentration of 10 µg / ml and incubated for 1 hour at 37 ° WITH. Then, a conjugate of highly specific goat antibodies to rabbit immunoglobulins with horseradish peroxidase in a 0.2% solution of gelatin in PBS is added to the wells. The plates are incubated for 40 minutes and the substrate for the enzymatic reaction is added (o-phenylenediamine in phosphate citrate buffer containing 0.012% hydrogen peroxide). After developing okra0

five

Skiing (20 minutes at 20 ° C) is stopped by adding 2N sulfuric acid solution to the wells. Evaluation of the reaction is carried out using a spectrophotometer with a vertical beam at 492 nm. According to the obtained values of optical density, calibration curves were constructed. The affinity of monAT was found to be sufficient to create an immunoassay test system for determining the concentration of TSH in the range of 0.4-50 mIU / ml in human biological fluids.

Claims (1)

Invention Formula The strain of hybrid cultured cells No. VSCC 366D of animals Mys musculus L is a producer monoclonal antibody to human thyroid hormone.