SU1414870A1 - Strain of hybrid cultivated cells of mus musculus animals used for producing monoclonal antibodies for ca two plus - atphase of sarcoplasmaticreticulum of dog heart - Google Patents

Abstract

The invention relates to biotechnology and can be used for cardiological research. The aim of the invention is to obtain a strain of hybrid cultured animal cells of the MUS musculus producing monoclonal antibodies (MonAT), which react with Ca-AT phase from the heart and skeletal minceps of animals and humans. The strain is obtained by hybridization of splenocytes of BALB / C mice immunized with the calcium-ocadate sarcoplasmic reticulum of the dog's heart muscle with P3-Ag 8 “653 myeloma cells. The cells are cultured in RPMI-1640 medium with 10% donor calf serum at 37 ° C in an atmosphere of 5% CO, the cells are passaged once every 3-4 days, the multiplicity is 1:10. The secretion of MONAT for 3-4 days of cultivation is 15-20 µg / ml of culture medium and 5 mg / ml of asci-. . tic fluid. MonATs belong to the Ig M class, they specifically interact with Ca - ATphase from the heart and skeletal mincers of animals and humans. MonAT can be used to diagnose myocardial and cardiovascular pathologies. vascular system, 2 tab.

Description

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; The invention relates to biotechnology and - i cardiological studies can be used. : The purpose of the invention is the production of ftam of hybrid cultured animal cells Mus musculus, producing monoclonal antibodies MonAT), interacting with the LT-phase of the sarcoplasmic reticulum — jjyMa (CP) of the heart and skeletal muscles in humans and the skeletal muscles. I Strain obtained as follows. The first BALB / C line is immunized with three peritoneum but with a calcium-skal preparation. CP of the dog's heart muscle. The first immunization was performed with IPO µg of antigen in Freund’s complete adjuvant. Repeated antigen at a dose of 5 pg in PBS buffer (0.15 M NaCl, 0.005 M Na-phosphate buffer, pH. 7.46) was administered intraperitoneally after 7 days, and then through H, 15, 16 and 17 days. The spleen skeletons are used for the death of rhodization on the 18th day.

f Myeloma cells of the P3-Ag8 653 pjacTHT line on RPMI 1640 medium containing 2 mM B-glutamine, 100 µg cannine in 1 ml, 0.05% mercaptoethanol, ai also 10% of donor or embryo-tel serum. The cells are cultured at 37 ° C in the CO -cot-. Fusion of spleen cells and | muni cells with M1 / 1 cells were performed using polyethylene glycol mol. 2000

A suspension of splenocytes is obtained from the spleens in a glass homogenizer and washed in a short-short medium Needle. Then, the cell suspension is centrifuged in percrlle (density), and cells deposited for 20 minutes at a speed of 2000 rpm are used for fusion, 50 million cells of the synovum are mixed with 10 million myeloma cells and centrifuged for 10 minutes at 900 rpm. The mixture of cells is placed in a thermostat at 37 ° C for 20 minutes. The supernatant was discarded, and 0.6 ml of warm 40% polyethylene glycol solution in RPMI medium with 15% dimethyl sulfoxide was added to the cells over 2 minutes. The cells, after fusion, are rinsed with RPMI medium, suspended in selective HAT medium containing 0.1 mM hypoxanthine, 16 μM thymidine, 0.4 μI aminopterine and 0.3 μM glycine. Fetch scattered in 96-lurochny flat-bottomed

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records from. calculating 50,000 cells per well. Hybridoma populations secreting antibodies to the CP membranes of the cardiac dog mint into the culture medium 5 are further cloned by the final dilution method. Peritoneal cells of the O BALB / C line are used as a feeding layer. The resulting strain denotes 1EZ, it is stored in the Specialized Collection of Transplanted Somatic Vertebral Cells of the Institute of Cytology, Academy of Sciences of the USSR, under

5 HACS (P) 96D and is characterized by the following features.

Cultural features. Standard cultivation conditions are RPMI-1640 hearts with 10% donor bones

0 serum, 4 mM L-glutamine

100 μg / ml kanamycin, 0.05 mM mercaptoethanol at 37 ° C in an atmosphere of 5% CO2.

Plastic containers are used to isolate the strain; A sowing dose of 100 thousand cells per 1 ml, the cells are passaged once every 3-4 days, the multiplicity of sieving is 1:10. Suspension culture. Cultivation in the body of animals. For balancing ascites, suitable BALB / C mice are suitable. Mice 7–15 days before the injection of strain cells were injected intraperitoneally with 0.5 ml of prisant. Fragile injected intraperitoneally

5 1-3x10 cells in 1 ml of Eagle medium. Ascites forms after 12-14 days. The productivity of the strain. The secretion of SonAT on the 3-4th day of cultivation is 15-20 µg in 1 ml of culture;

Oral medium and 5 mg in 1 ml of ascites fluid when determined by radioimmunoadsorption inhibition method.

Characteristics of the resulting product. The strain produces NonAT, attributed to class 1g. Method for assessing specificity: a test for the binding of MonAT to immobilized CP membranes or to purified Ca -ATphase (radioimmunoassay). Antibodies

 do not have an inhibitory effect on Ca-AT phase activity.

Product stability MonAT production is maintained for at least 20 in vitro passages and up to three

g ascitic passages.

Cryoconverting. For long-term storage, strain cells are frozen in fetal bovine serum with the addition of 10% dimethyl.

sulfoxide. Freezing mode: 40 minutes to +4 ° C, then 1 / min to -40 ° C. After freezing, the cells are transferred to liquid nitrogen. Thawing is carried out in a water bath at +37 C. Cell viability after thawing is 70-80% by trypan blue staining.

Contamination Bacteria and fungi in culture were not detected during long-term observation and culture on nutrient media. Microplasma infection was not detected by staining with dyes on DNA and the incorporation of the thymidine label.

Example. On 96-cell flexible PVC sheets, I sorb the purified Ca-AT phase - 2 μg per well in 100 μl PBS buffer. After 2 hours of incubation at room temperature, the wells are washed with PBS containing 0.05% Tween 80 (buffer A). After washing three times, the well is filled with buffer A and incubated for 1 hour at room temperature or overnight. For testing, 100 µl of the culture fluid of the hybridoma clone is added to the well, followed by 1 hour of incubation and washing with buffer A. Thereafter, the well is added to the well. 100 µl of the same buffer containing the developing reagent - labeled J rabbit antibodies to the light chains of mouse immunoglobulins (100 thousand cpm). After 1 h of incubation, the plates are washed and the cells are counted in a gamma spectrometer.

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The specificity of the reaction of monoclonal antibodies 1EZ with Ca-AT phase is shown in Table. one.

The results presented in table. 1, testify to the high specificity of the obtained MonAT to the Ca-AT phase of the dog's heart.

Data on the ability of MonAT 1EZ to react with the CA -ATphase PC of the heart and skeletal muscles, as well as the erythrocyte cell membrane, is given in Table. 2

The results are shown in Table. 2, indicate that the resulting hybridoma strain produces MonAT, reacting with the CA -ATphase from the heart and skeletal mice in a number of laboratory animals, as well as from human heart and erythrocytes. Thus, the obtained MONAT differ from the known ability to cross-react with the CA-ATphase of the CP of the heart, as well as

human erythrocytes. This allows the use of MonAT data to diagnose pathologies of the micromyocard and cardiovascular system.

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Claims (1)

Invention Formula A strain of hybrid cultured 4 animal cells Mus musculus, BCKK (II) No. 96 D, used to produce monoclonal antibodies to the αATphase of a dog's sarcoplasmic reticulum. I T a b l and c a 1 Sa-ATphase heart dogs Strain 1EZ. Also Bovine serum albumin (control) Sa-ATphase dog heart Also 6300 1:10 5900 1: 100 4400 1600 450 400 360 14148706 Table 2 Antigen (drug from which Ca-ATphase is released Guinea pig heart muscle Heart dog Rabbit heart muscle Human heart muscle Skeletal dogs Skeletal Rabbit Mice Skeletal rats Erythrocyte man Bovine serum albumin (control Binding of labeled second antibodies, counts / min 8050 8090 6640 6230 8850 11550 10190 6280 450