SU1497214A1 - Strain of hybrid cultivated cells of animals mus musculus - producer of monoclonal antibodies to ca2+/mg2- dependent on endonuclease of nucleus of manъs spleen lymphocytes - Google Patents

Abstract

This invention relates to hybridoma technology and can be used in experimental and clinical medicine. The purpose of the invention is to obtain a hybridoma producing monoclonal antibodies to CA ²⁺ / MG ²⁺ -dependent endonuclease of human spleen lymphocytes. The DS strain is obtained by fusion of spleen cells of BALB / C mice immunized with an extract of cellular nuclei of human spleen lymphocytes containing the CA ²⁺ / MG ²⁺ enzyme dependent endonuclease and myeloma line X63.AG8.653. The strain deposited under the number UASK (P) N117D and produces antibodies JGG class ₂ . Antibody secretion on day 4 of cultivation was 10-20 µg / ml of medium and 1-3 mg / ml of ascitic fluid. 3 tab.

Description

The invention relates to hybridoma technology and can be used in experimental and clinical medicine.

The purpose of the invention is to obtain a hybridoma producing monoclonal antibodies to Ca / Mg-dependent endonuclease of human spleen lymphocytes .-,

Strain was prepared as follows.

The strain is a product of the KnevoK spleen fusion of BALF / c mice and myeloma line X63.Ag8.653. Mice are immunized with an extract of human cell lymphocytes of the human spleen containing the enzyme Ca / Mg -dependent endonuclease. At the same time use two schemes of immunization. -.

The first scheme. In the BALB / c line, males weighing 18–20 g are immunized subcutaneously at 5 points using complete Freud's adjuvant at a dose of 100 μg of protein per mouse. After 10 days, the mice are immunized again intraperitoneally at a dose of 100 µg, another 10 days later, 200 µg of protein is intravenously administered within 6 months. The mice rest, then they are immunized intravenously at a dose of 100 µg of protein per mouse, and a week later the resolving immunization is administered to the intrasilenosis at a dose of 100 µg of protein. On day 3, spleen cells immunized take on hybridization.

The second scheme, the same line is immunized twice with glue extract

14U721A

accurate der lymphocytes of the spleen cheoveka with an interval of 7 days. The antigen is introduced into the intracellular membrane at a dose of 100 µg for protein, Hybrid limy,. of human spleen cells with myelomas of the spleen and carried out on

4 stops For fusion, cells using X63Ag8.6535 cells that are in the oarithmic growth phase are used. Mice d are sacrificed with a cervical daxlocation, the spleen is aseptically removed, and the cell suspension is prepared by homogenization in a Potter homogenizer,

Cell fusion is carried out as follows.

Suspension of myeloma cells and lymphocytes is mixed in a flat-bottomed glass vial in the ratio X). 1IOo. The vial is centrifuged 200 g.

5 min, the supernatant is removed and 2 ml of a 50% aqueous solution of plastic glycol (M, B, 1500) is added dropwise to the sediment of the cell. Cells are suspended with light-CMM-25 by shaking the vial. Through

1 min in a vial add 10 ml of DMEM medium, then for 3 min more

50-60 bp The bottle is centrifuged

200 g 5 MHHj remove the supernatant and 30

cell pellet resuspended in 60 ml

medium DMEMj 15% fetal serum.

The cells are incubated in a vial at

37 ° C 6-12 h. After incubation, the medium

in vials replaced with selective

DMEM-GAT medium; containing, gentamicin 80 mg / No. 1, giioksantin 5-10-3 aminopterin, thymidine 8-10 M.

The cells at a concentration of 2-310 / ml are distributed in 96-well wells: 40 shtans, the bottom of which is precoated with a feeder layer of syngenic peritoneal macrophages (6 x 10 cells per well). Cells were cultured in plates at 37 C in the atmosphere of 5% CO. and 96% humidity. The medium is changed every 3 days at 50%. Mass cell death is observed at 3-6 days of cultivation on selective medium. The growth of hybrids is observed at 5-7 days. The first testing of supernatants was carried out on day 10 of culture after hybridization. Hybrid cells are cultured on UMEM medium with the addition of 15% fetal serum, gentami-, hypoxanthine cicadas, and an amino-tester of Chudin by-bye for 30 days, then for THAT days

 DMEM with 15% fetal serum.,

genta No- cyn, hypoxanthine, thymidine. Further cultivation is carried out on DMEM medium with 15% fetal serum and gentamicin.

Hybridoma cloning is carried out by the method of limiting dilutions.

As a result of testing supernatants from 200 cultures obtained by fusion of myeloma with lymphocytes of mice immunized according to the first scheme, 8 positive lines of hybridoma cells I and Iio were obtained for the second scheme of immunization - 13 positive clones from 200 primary cultures. All stable positive lines are cloned and frozen in liquid nitrogen. Clone DS cloned 5 more times. In antibody production, clone DS is cultured under in vivo conditions in the form of ascitic fluid in the abdominal cavity of syngeneic mice.

The strain is deposited under VSKK (P) number 117D and is characterized by the following features.

Cultural features.

Standard cultivation conditions. The culture medium is Dulbeco Medium supplemented with 10% fetal bovine serum, 4 mm L-glutamine. 80 μg / ml gentamicin, 37 C. 5% COgj 96% humidity. The use of Eagle's medium is acceptable, with the addition of 15% bovine serum.

Glass or plastic dishes are used to grow the strain of the strain. 240 -10 cells of DS strain were introduced into a 25 ml vial in 5 ml of medium. Passage 1 time every 3-4 days with the same dose of cells. The multiplicity of sieving 1: 4

Cultivation in the body of the animal. To grow a tumor from a strain, a BALB / C lower line is used. Freshly for 7–30 days (optimally 14) days before the injection of the strain, 0.5 ml of the bailer is injected intraperitoneally, then injected in 3, cells per mouse in 1 ml of Eagle's medium. Ascitic fluid is harvested for 10–15 days. From ascites, they are intertwined on fresh 1-540 cells per mouse, 4-6 passages.

Characteristics of the resulting product. Monoclonal antibody (monAT) refers to IgG, a mouse, fixing protein A. Capable of binding Ga / Mg -dependent endonuclease of human cell nuclei

51497214

endonucleases of peripheral blood lymphocytes of humans and cattle, rat hepatocytes, n. Lesbians of CBA, C57B / C and BALB / C mice. MonAT reacts with endonucleases of cell dermatothymocytes of С57В / С and СВА lines and do not react with thymus endonucleases of BALB / c mice.

6

Pic polzov a.chk c strain l n.-i .-; i icTp; i is the following example.

Example. The 96-well plates of the plate are coated with protein A, followed by the test samples of the culture medium of strain DS. After reacting monat with protein A, the wells are washed and cell extract is added.

In chronic lymphocytic leukemia of large io ix der human silenocytes or

bovine antibodies bind the spleen lymphocyte endonuclease to a lesser extent, and the peripheral blood lymphocyte enzyme is more than normal. Antibodies do not react with bovine pancreas DNA 1, human alkaline forphatase, and human blood proteins.

Productive ability. The secretion of monat on day 4 of cultivation was 10–20 µg in 1 ml of medium and 1–3 mg in 1 ml of ascitic fluid. Antibody synthesis is assessed by heterogeneous immuno-enzyme analysis.

The stability of monAT production is maintained for 30 passages in culture and 5-6 passages in animals if the hybridoma is transplanted.,

Cryoconservation Cells of the strain to the resuspender: are in fetal bovine serum containing 12.5% dimethyl sulfoxide, in concentrated 3-4–10 µl / ml, poured into plastic ampoules at 4 C. Then the cells are frozen according to the program - until

-40

about

with

decrease and then

temperature ya 1 / min, and then to -196 ° C at -10 ° C / min. Defrosting: 1 min. The cells were diluted in 10 ml of complete medium and transferred to a culture flask. Cell viability on trypan blue incorporation is 75-90%.

Contamination Bacteria and fungi in culture were not detected during long-term observation and culture on nutrient media.

purified extract of Ca / Mg -dependent endonuclease. After the monat-bind enzyme, the supercoiled DNA plasmid is added.

as substrate for the enzyme. A positive reaction is determined by the hydrolysis of the super coiled Ami; plasmids after electro-theoretical analysis of JlJiK cleavage products

Ca / Mg-dependent endonuclease, fixed monat.

MonAT DS can be used as a quantitative determination. Ca / Mg-dependent endo-nuclease,

and to determine its activity of the E complex antigen-antibody B tissue extracts and biological fluids.

Antibody Binding Results

the DS strain with purified Ca / Mg i -z Bi with the STEM endonuclease following;

Antibodies

Activity

/ Mg

dependent endonuclease adsorbed to antibodies ;;.%

100 89 44.5

DS

DS 0.1 DS 3.1 Cattle Serum

45

The results of the binding of DS strain antibodies with various lucleases are shown in the table.

The binding of the DS strain antibodies to Ca / Mg-dependent endonuclease of extracts of human lymphocyte cell spleen extracts is 100%, 10% mice and 45% cattle.

The use of the strain makes it possible to obtain a high specificity monaton of the Ca VMg-dependent endonuclease of human lymphocyte cell lymphocytes, which can be used both for the quantitative and qualitative determination of the endonuclease as well as for histochemical research of enzyme isoforms associated with the study of their intracellular localization, study the distribution of farmfeit isoforms in human lymphoid organs, the determination of activity and the content of dependent enzyme in lymphoproliferative diseases.

b

formula of invention

The strain of hybrid cultured animal cells Mus musculus VSCC (P) No. 117D is a producer of monoclonal antibodies to the dependent endonuclease of human cellular lymphocytes of the human spleen.

Claims (1)

Claim The strain of hybrid cultured animal cells Mus musculus BCCC (P) No. 117D is a producer of monoclonal antibodies to Ca ²⁺ / Mg ²⁴ - dependent endonuclease of the cell nuclei of human spleen lymphocytes.