SU1497213A1 - 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. The purpose of the invention is to obtain hybrid cultured animal cells that produce monoclonal antibodies (monAT) to the CA ²⁺ / MG ²⁺ -dependent endonuclease of human lymphocyte cell lymphocytes. The strain was obtained by fusion of spleen lymphocytes of BALB / C mice and X63 myeloma. AG8.653. Mice are immunized with an extract of human cell lymphocytes of the human spleen containing the enzyme CA ²⁺ / MG ²⁺ -dependent endonuclease. The strain deposited under the number VSKK (P) N116D. MonAT secretion on the 4th day of cultivation 10-20 µg in 1 ml of medium and 1-2 mg in 1 ml of ascitic fluid. MONAT JGG ₂ mouse. The stability of antibody production is maintained for 30 passages in culture and 10 passages in animals if the hybridoma is transplanted. 2 tab.

Description

The invention relates to hybridoma technology and can be used to obtain monoclonal antibodies (monAt) to Ca / Mg-dependent endonuclease of human lymphocyte cell lymphocytes.

The purpose of the invention is to obtain hybrid cultured cells of animals producing monat for the dependent endonuclease of human dermal cell lymphocytes.

Strain was prepared as follows.

Strain - product of lymphocyte fusion of spleen of BALB / c mice and myeloma

X 63.Ag8.653. Mice are immunized with an extract of human cell lymphocytes of the human spleen containing the enzyme Ca - / Mg-dependent endonuclease, and two schemes are used for immunization.

first scheme. Mice of the BALB / c line, males weighing 18–20 g, are immunized subcutaneously at 5 points using Freund’s complete adjuvant at a dose of 100 μg of protein per mouse. After 10 days, the antigen is administered intraperitoneally in incomplete Freud's adjuvant at a dose of 100 µg of protein per mouse, then immuni

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The dose is administered intravenously.

200 µg of protein. For 6 months, the animals rest, then 100 µg of extract are injected intravenously, after 7 days the immunization is repeated in the whole glen in a dose of 100 µg of extract protein and on the third day after resolving immunization, the spleen cells are taken for hybridization,

The second circuit. Mice of the same line are immunized twice with an extract of cellular nuclei of human spleen with an interval of 7 days. The antigen is administered intra-splenically at a dose of 100 µg per protein. Hybridization was performed on day 4.

In hybridization, myeloma cells 3 P3, X63, Ag8.653, nonclassifying immunoglobulins, which are in the logarithmic growth phase, are used.

The rag is sacrificed, the spleen is removed aseptically, and the cells are suspended in a homogenizer.

A suspension of myeloma cells and spleens of immunized mice are mixed in a flat-bottomed vial in a ratio of 1:10. The vial is then centrifuged for 200g for 5 minutes, the supernatant is removed and 2 ml of a 50% polyethylene glycol solution is added to the precipitate dropwise (M ; B, 1500). The cells are suspended by shaking the vial gently. After 1 min, add 10 ml of DMEM medium to the vial, then another 50-60 mp for 3 min. The vial is centrifuged at 200g for 5 minutes, the supernatant and resuspended cell pellet are removed in 60 ml of DMEM medium, 15% fetal serum.

The cells are incubated in a vial for 6-12 hours. After incubation, the medium in the vial is replaced with a selective one having the following 1st composition: DMEM medium, 15% fetal contraction, hypoxanthin M, aminopterin 2 10 M, thymidine 8 10 m, gentams 80 mg / ml qing (DMEM-GAT medium),

The cells at a concentration of 2–310 cells / m 1 are distributed across the wells of flat-bottomed 96-well plates, the bottom of which is preliminarily covered with a syngeneic feeder layer. peritoneal macrophages (cells per well), the cells are cultured in plates at 37 ° C in an atmosphere of 5% CO2, and at 96% humidity.

The medium is changed every 3 days for 50%. Mass death of myeloma cells is observed, starting from 3 days of cultivation, the growth of hybrid colonies is increased

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5 o

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begins on day 5-7 of cultivation. Testing of the supernatants was performed starting from the 10th day of culture after hybridization. Hybrid cells were cultured on DMEM-GAT medium for 30 days, then 10 days on DKEM-GT medium (hypoxanthine 5-10-3 thymidine 8-10 M), after What hybridomas are cultivated on VGM medium, 15% fetal serum.

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

As a result of testing the supernatants from 200 primary cultures obtained by fusing myeloma with the lymphocytes of mice immunized according to the first scheme, 8 stable positive lines were obtained; the second immunization scheme - 13 positive lines from 200 primary cultures. All lines are cloned and frozen in liquid nitrogen. The clone DN was cloned 5 more times; for the production of antibodies, the clone DN is cultured under in vivo conditions in the form of ascites fluid in the abdominal cavity of syngeneic mice,

The resulting strain DN is deposited under the FCC number (P) No. 116D and is characterized by the following features,

Cultural features.

The standard cultivation conditions are Dulbeko's medium with the addition of 10% calf embryo, 4 mm of L-glutamine, 80 µg / ml gentamicin, 37 ° C, 5% CO ,, 96% humidity, use of Kgl medium with 15% added serum of cattle.

Glassware or plastic dishes are used to isolate the strain. In a vial with a volume of 25 cm in 5 ml of medium, make cells DN, Passage 1 time every 3-4 days with the same dose of cells, the multiplicity of sieving 1: 4.

Cultivation in the animal.

BALB / c lines are suitable for growing a tumor from a strain. Fresh for 7–30 days (optimally 14) days before the injection of the strain is administered intraperitoneally 0.5 ml of pristan. Gitamm is injected intraperitoneally at 3.2x10 cells per microspider in 1-ml Eagle's medium. The ascic fluid is collected as it occurs (10-15 days). From ascites, the strain is transplanted in fresh mice with 1-5x10 cells per mouse, 4-6 passages.

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nom observation and crops on nutrient media.

The use of the strain is illustrated by the following example.

Example. To determine the presence of specific antibodies in cynef-natanta, hybridomas use flat-bottomed plates, the magnifying glasses of which are human predocytes or purified preparatively treated with protein A, Ca / Mg-DNA-ase, are added. After washing the plates, 50 tons of strain phosphorescence is added.

MonAT secretion on the 4th day of cultivation 10-20 µg in 1 ml of medium and 1-2 mg in 1 ml of ascitic fluid. Method for assessing the synthesis of antibodies - heterogeneous enzyme-linked immunosorbent assay: plastic coated with protein A, treated with an extract of cellular nuclei

supersperate DNA of plasmid pBR 322 A positive reaction is determined by the hydrolysis of the superspirate form of the plasmid after electrophoretic analysis of the DNA cleavage products of the / / Mg-dependent endonuclease.

The stability of antibody production is maintained for 30 passages in culture and 10 passages in animals if the hybridoma is transplanted.

Characteristics of the product, Monoclonal antibody IgG i-ibi, Monoclonal antibodies are capable of binding Ca / Md-dependent endonuclease of human cellular splenocytes endonuclease dermal lymphocytes, human peripheral blood and cattle, hepatocytes of rats, spleen of CBA mice , C57BL / 6 and BALB / c. In addition, monat reacts with endonucleases of cell dermatocytes of the BALB / c line and does not react with thymus endonucleases of C57BL / 6 mice and CBA. In chronic lymphocytic leukemia of cattle cattle monat bind the spleen lymphocyte endonuclease to a greater extent, and the lymphocyte enzyme of the peripheral lymphocytes less than normal. These antibodies do not react with bovine pancreas DNA I, human alkaline phosphatase, and human blood proteins.

Cryopreservation, the cells of the strain are resuspended in fetal body serum containing 12.5% dimethyl sulfoxide, at a concentration of 3-4x10 cells in 1 ml, poured into plastic ampoules with, then the cells are frozen according to the program until the temperature decreases by 1 C / min and then up to -196 ° С at Yu C / min,

Defrosting: 1 min at 37 C, Cells

diluted in 10 ml of complete medium and transferred to the culture flask.

Contamination Bacteria and fungi in culture are not detected for long

with test buffer (PBS) and treatment with 1% bovine serum albumin, the test samples are added to the wells

5 and incubated overnight at

0–4 ° C. The wells are washed 4–6 times with PBS, 50 μl of a human cell splenocyte extract with a protein concentration of 5 mg / ml is applied and incubated

0 bh at 0–4 ° C. The wells are washed 2 times with PBS and 2 times with 10 mM Tris buffer, pH 8.0, 10 mM MgCl1, 1 mM CaC (IB), then the PDK substrate is super-helical form of the plasmid pBR

5 322 at a concentration of 0.6 µg / ml in IB and incubated for 2 hours at 37 C.

Plasmid DNA digestion products are divided into 1.2% agarose blocks. When applying cuts on the substrate

The DNA supercoiled plasmid transforms into open circular and linear plasmas, which differ from the supercoiled form in electrophoretic mobility. Thus, the presence of antibodies to the / / Mg2-dependent endonuclease is judged by the disappearance of supercoiled DNA and the appearance of the open circular and linear forms of the plasmid pBR 322,

The obtained DN antibodies interact with the purified enzyme / Mg2 - - - dependent endonuclease of human cell splenocytes.

The results of the binding of antibodies DN 5 to purified Ca / Mg -dependent human splenocyte endonuclease are as follows:

The activity of dependent endonuclease sorbed on antibodies of the DN line,%

44.5.

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Antibodies

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DN Serum cattle

cattle

ABOUT

During sorption on antibodies of human cell spleen extracts containing Ca / Mg-dependent endonuclease in the antigen-antibody complex, the enzyme exhibits properties similar to purified Ca / Mg-dependent endonuclease. In particular, the enzyme activity in the presence of no more than 80% of the activity in the presence of Ca and no more than 32% of the activity in the presence of Ca and.

In order to determine the species specificity of antibodies, the lines examine their binding to the Ca / Mg2-dependent eudonuclease of the cellular spleen nuclei of humans, cattle and BALB / c mice,

Binding of DN line antibodies to / Mg-dependent endonuclease. extracts of cellular dermal lymphocytes of human spleen 100%, mice 55% and cattle 37%. The results show that DN antibodies are species specific.

According to this, although an enzyme of evolutionary distant species can also bind the stomach -) .-. of

In addition, the binding of DN antibodies to various components of the cell core — DNA, DNP, RNA, and the histone fraction was studied. In no case was the binding of antibodies to the named components detected.

The use of the invention allows to obtain monat against the Ca / Mg-dependent endonuclease of human lymphocyte cell lymphocytes.

Claims (1)

Invention Formula The strain of hybrid cultured animal cells Mus musculus VSCC (P) No. 116D - producing monoclonal antibodies to Ca / Mg -dependent endonuclease of human lymphocyte cell lymphocytes.