SU1514770A1 - Strain of cultivable hybrid cells of mus musculus as producer of monoclonal antibodies against factor v of erythrocytes of cattle - Google Patents

Description

The invention relates to biotechnology and can be used „in agriculture in the determination of blood groups of cows in immunological studies on farm animals in commercial and livestock breeding. The aim of the invention is to obtain a strain of cultured hybrid cells producing monoclonal antibodies (mAbs) against the ν-antigenic factor of erythrocytes of cattle with high titer. Strain producing ICA against cattle V-antigen factor in bovine erythrocytes was obtained by hybridization of mouse myeloma cells with spleen cells of a mouse immunized with bovine erythrocytes. The resulting hybrid cells were selected on the basis of the secretion of ICA to ν-antigenic blood factor of cattle. The selected cell plan was characterized in many ways and as

The strain was deposited in the cell culture collection of the Institute of Cytology of the Academy of Sciences of the USSR - VSKK under No. 56 Ts.

1514770 A1

animals in commercial and livestock breeding.

The purpose of the invention is the obtaining of a strain of cultured hybrid cells,

producing µa against the ν-antirenic factor of erythrocytes

large horns1) 14770

That lamb with a high titer.

II p and me 1. Producer cells

ICA (the mouse chromosomes are Mpz tyzsi1i5. In the vast majority of cells I meet ·; ’two marker chromosomes: a small metacentric M,

(60% of cells) and a large submetacentric M _g (70% of cells). In single cells (2-10%) there are 6 arms of different types with respect to the ratio and differential staining (M _L –M ₈ ).

Physiological signs - features of the cultivation of the strain.

The strain was cultured on a growth medium in Dulbecco's modified Eagle's (DMEM) supplemented with 10% fetal bovine serum and 1tM nonbasic amino acids, sodium pyruvate 1tpM, 2TM glutamine, 5x10 nT ⁵ M 2-mercaptoethanol and 40 units / ml geptamitsina.

Cultivation is carried out at 37 ° C in an incubator with a content of 5-8% C0 _£ or in sealed vials in a thermostat without CO 2 supply. The method of cultivation - suspension. The multiplicity of sieving is 1: 3 when reseeding after 48 hours. Thus, the sowing dose of cells is 150 thousand cells in 1 ml, and the maximum population density of cells is 600 thousand cells in 1 ml.

When growing on the indicated medium, the cells of the strain have a rounded shape. The population doubling time is 36 hours. Cell cloning is carried out in 96 well plates using mouse microphages as a feeding layer. When seeding 3-4 cells per well, the cloning efficiency is 70%.

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Cryoconservation is carried out in serum of cow embryos with the addition of 10% dimethyl sulfoxide (0M50) at a concentration of 1 x 10 cells in 1 ml. Cell viability of the strain after thawing is 75-80%.

Inoculation of cells of strain 56ϋ syngeneic mice causes an ascites tumor to form, accompanied by the release of ascites fluid in an amount of 1–8 ml. Ascitic fluid contains specific µa to the ν-antigenic factor of cattle blood.

The ability to produce ICA cells of the strain 56Ώ is maintained for 3 months of continuous cultivation of ίη νίϋΓο cells. After freezing the cells, they retain the ability to produce MCL. Graft

Mice of cells at different passages of cultivation consistently cause the formation of ascites tumors in mice with a high titer of µA in ascites fluid.

Characteristics of the mAb produced by the strain 561).

The specificity of the obtained µa is determined using standard serological hemolysis test. The analysis is carried out on a panel of erythrocytes, consisting of 82 blood samples of cattle, in which the antigenic structure by blood group was determined using monospecific allo-antisera. The obtained mAbs exhibit specificity similar to monospecific anti-ν serum.

To isolate MCA from ascitic fluid, immunoglobulins are precipitated with ammonium sulfate to 50% saturation. The precipitate is separated by centrifugation and the analysis of the ICA. According to the data of electrophoresis in polyacrylamide gel with sodium dodecyl sulfate, MCA belongs to class C immunoglobulins. The molecular weight of the heavy chain of immunoglobulins is 58-60 κϋ, and the light chain is 26 κ.

The productivity of cells of the strain 56ϋ.

By cultivating the cells of the ίη νίνο strain in Ba1b / s mice from one mouse, 2-8 ml of ascites fluid (4 ml on average) can be obtained.

The antibody titer in ascitic fluid, established by hemolysis of red blood cells with cattle micromethod, is 1: 2048. However, storage of ascitic fluid for 6 months at 20 ° C, triple freezing and thawing of it cause a decrease in antibody titer by 100 times.

Example 2. To obtain ICA, strain 56ϋ cells are inoculated with Ba1b / s syngeneic mice. Take 10 mice. Each mouse is pre-administered with 0.5 ml of bailiff. After 10 days, the animals thus prepared are inoculated with 2 · 10 -4 · 10 hybridomas

cells in a volume of 0.5 ml of saline. After 10-14 days, the mice are sacrificed and ascites fluid is removed from the abdominal cavity. A total of 10 mice received 20.5 ml of ascitic fluid. Cells are separated by centrifugation (10 min, 2000 rpm),

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and the supernatant is used

as a source of ICA (reagent ΜΚΑ-ν).

The presence of specific mAbs. In ascitic fluid is determined by the hemolysis reaction of red blood cells.

To do this, first prepare individual suspensions of erythrocytes of cattle: blood from the jugular vein of cattle is collected in test tubes with a standard anticoagulant solution; red blood cells 4. times washed with a 0.08% solution of) NaCl by sequential centrifugation and dilution (11) minutes,

2000 rpm), prepare a 2.5% suspension - | ziyu of erythrocytes in a 0.8% solution of DaCl. Then, in 96-well immunological plates, 20 ml of ascites fluid and 20 ml of erythrocyte suspension are mixed. The resulting 2 mixture was incubated at 37 ° C for 30 minutes and 50 ml of rabbit complement was added, then incubated again for 1 hour at 37 ° C and hemolysis was evaluated using a four-ball system. 2 Use the red blood cell suspension of cows. Animals previously characterized by antigens groups

of blood with the help of monospecific allo-antisera. The analysis shows that the obtained ICA cause hemolysis of only those erythrocytes, which, along with others, carried antigenic factor V and did not react with erythrocytes that did not contain antigenic factor V.

To establish the antibody titer of the reagent ICA, ν-ascitic fluid is diluted with 0.08% NaCl solution and the maximum dilution is determined at which it is even more capable of causing hemolysis of red blood cells of the cattle. For this purpose, use the specified method of setting the reaction of hemolysis.

It was established that the obtained ICA causes hemolysis of erythrocytes bearing antigenic factor V, up to a dilution of 1: 2048.

Claims (1)

Claim The strain of cultured hybrid cells of the animal Miz ps8si1i5 VSKK (II) No. 56ϋ - producing monoclonal antibodies against factor V of erythrocytes in cattle.