SU1638160A1 - Strain of hybridous cultured mammalian cells musculus, l- producer of monoclonal antibodies against of general antigen of acute lymphoblast leukosis cells - Google Patents

Abstract

This invention relates to hybridoma technology and can be used in the diagnosis of acute lymphoblastic leukemia (ALL). The purpose of the invention is to obtain a strain of hybrid cultured cells producing monoclonal antibodies (monAT) to a common antigen of ALL cells (OAG-ALL) that do not react with blood granulocytes. The strain is obtained by hybridization of myeloma cells RZ X63.Ag8.653 with splenocytes of BAIB / C mice immunized with the cells of the peripheral blood of a patient with lymphoid blast crisis of chronic myeloid leukemia. MonAT is selected by interaction in an indirect reaction of surface immunofluorescence with the leukemic cells of the same patient. The strain is cultivated on the environment DMEM with 15% serum of the embryo of the cow. 2 mM glutamine and 0.1 mg / ml gentamicin. The cells are passaged after 2-3 days. The strain is grafted onto syngeneic mice as ascites tumors. The strain produces monAT class lgC3. The titer of monat in supernatants 50, in ascitic fluids 10-10. The strain deposited under the number VSKK (I) No. 69 D. (L S

Description

This invention relates to hybridoma technology and can be used in the diagnosis of acute lymphoblastic leukemia (ALL).

The purpose of the invention is to obtain a strain of hybrid cultured cells producing monoclonal antibodies (monoAT) to a common antigen of ALL cells (OAG-ALL) that do not react with blood granulocytes.

Strain receive the following method.

Hybridization of murine myeloma cells RZH63 Ad8.653 and spleen cells of mouse BAIB / 3, 3-fold immunized with 2-week intervals of the cells of the peripheral blood of a patient with lymphoid variant of blast crisis of chronic myeloid leukemia. Leukemia cells are administered in an amount of 2,107 into the mouse tail vein. On the 3rd day after the last immunization, the spleen cells of the mouse and the myeloma RZX63 Ad8.652 were fused. Under sterile conditions, the spleen of the immunized mouse is removed and the cells are squeezed out in a Potter glass homogenizer. The cell suspension is filtered through 2 layers of gauze. Lymphocytes are washed three times with medium 199 and merged with myeloma cells in a ratio of 80 x 10b lymphocytes to 5 x 10b

about

SA) 00

ON Oh

myeloma cells. The mixture of cells is placed in a 100 ml vial in a volume of 20 ml of medium 199. In this vial, the cells are centrifuged at 800 rpm for 3 min and incubated for 20 min at 37 ° C. The medium is sucked dry and 3 ml of 50% polyethylene glycol, mol%, is added to the cells. 1500. After 75 seconds, 20 ml of medium 199 are introduced into the vial. The cells are washed 3 times, without agitation, and incubated at 37 ° C for 5 hours in 20 ml of GAT / DMEM selective medium containing 20% fetal embryo serum, 2 mM glutamine, 0.1 mg / ml gentamicin, 0.0136 mg / ml hypoxanthine, 0.000151 mg / ml aminopterin, and 0.00385 mg / ml thymidine. The cells are then poured into 0.2 ml in the wells of a 96-well flat-bottomed plateau. The medium is changed after 2-3 days. Colonies occur after 25 days in 96 of 96 holes. The selection of the hybrid-producing species is carried out in an indirect surface immunofluorescence (RIF) reaction on the patient's cells used for immunization. Positive reaction in 1 of 96 wells. Then the supernatant of this hybrid is tested on the blood cells of healthy donors. Cells from the well G 4 are double cloned by limiting dilution method on a feeder from thymocytes and splenocytes. When first cloned, colonies grow into 8 out of 12 holes. Supernatants in all of them react in FTA with the cells used for immunization. In the second cloning, the colonies were isolated in 5 of 12 wells, also all positive in RIF with patient cells used for immunization.

The resulting strain is designated ICO-35.

The strain of hybrid cells was deposited in the specialized collection of transplanted somatic cells of vertebrates of the All-Union collection of cell cultures under the number of VSKK (II) 69 D.

Cultural properties.

Plastic or glass utensils are used to grow the strain. In a vial of 45 cm3 in 5 ml of the medium, 5,105 cells of the strain are poured. Passage 1 time every 2-3 days with the same dose. The strain is grown at 37 ° C on MEM medium in Dulbecco's modification, containing 15% fetal calf or deer serum, 2 mM glutamine, 0.1 mg / ml gentamicin.

Antibody titers are determined by indirect surface immunofluorescence reaction on living blood lymphocytes. As the source of antibodies using culture fluid. The antibody titer in the culture fluid 1:50,

BAIB / C female mice are suitable for growing the strain as an ascites tumor. Intact mice 7–14 days prior to inoculation of a tumor, intraperitoneal irritant (Vaseline) was injected, strain cells were injected intraperitoneally with 107 cells per mouse in 4.0 ml of medium. After 7-14 days, mice develop ascites tumors. The volume of ascites from one mouse is 2-3 ml. The titer of antibodies in ascitic fluid is 104-105.

The karyological characteristic, the modal number of chromosomes is 85. Marker chromosomes have not been identified.

Productivity. Antibody production is maintained for 25 in vitro and 15 in vivo passages. The antibody titer in the culture fluid 1:50, in ascitic fluid - 10.

Contamination Bacteria and fungi in culture were not detected during long-term observation and when sowing on nutrient media. Infection with mycoplasma was not detected by staining with dyes on the DNA and thymidine label of the culture medium.

Cryoconservation Cells of the strain are resuspended in DMEM medium containing 15% calf fetal or deer serum. 10% DMSO is added to the cell suspension. Cell concentration (1-2) x 10 ml. Ampoules with cells are placed in liquid nitrogen. Defrost at 40 ° C for 70 s. Cell viability after thawing 80-90%.

Characteristics of the target product.

Strains of hybrid cultured cells IKO-35 produces monat class AdbZ. The specificity of mono ICO-35 is determined in an indirect reaction of surface immunofluorescence using a standard procedure.

ICO-35 monoclonal antibodies do not react with mononuclear blood of healthy donors, granulocytes. T- and B-lymphocytes, erythrocytes and platelets. In the bone marrow of healthy people, monat ICO-35 is determined from 0 to 9% of antigen-positive cells. In the thymus of children monat ICO-35, from 0 to 5% of antigen-positive cells are determined. At the same time, mono-ICO-35 reacts with all OAG-ALL-positive blast cells of patients with ALL and chronic myeloid leukemia in the stage of an imperious crisis. Monat ICO-35 reacts with OAG-ALL-positive pre-B cell line Reh-b and does not bind to cells of the lines K-562, Jurkat, Prise, HL-1, Molt-4, ML-1. Comparison of the reactivity of the monocat ICO-35 and monat I 5 proves their identical reaction with the cells of patients, but they

differ in reaction with blood granulocytes,

PRI me R 1. A child suffering from acute lymphoblastic leukemia (ALL) is a subtype according to the FAB-classification L 1.

Immunological phenotype of regional bone marrow cells:

ICO-1 (1a-antigen83%);

ICO-01 (undifferentiated blasts antigen) 1%;

ICO-10 (early thymocyte antigen) 0%;

ICO-11 (LFA-1 antigen) 0%;

ICO-GM1 (myeloculocyte antigen) 7%;

ICO-G2 (x-hapten) 3%;

NAEZ - (glycophorin A) 0%;

NAE9 - (erythroblast antigen) 0%;

ICO-20 (SD38 antigen) 8%;

ICO-15 (E-receptor antigen) 68%;

T5D (T cell antigen) 1%;

SD7 (T cell antigen) 4%;

SD 10 (OAG-ALL-antigen) 97%;

IKO-35 (OAG-ALL - antigen) 95%;

Sig (surface immunoglobulins) 2%;

Cylg (cytoplasmic immunoglobulins) 7.5%.

Immunological diagnosis - common ALL.

PRI mme R 2. Child, a patient with ALL, a subvariant by FAB-classification L1.

Immunological phenotype of blast cells.

PID-1 (1a-antigen) 76%;

ICO-10 (early thymocyte antigen) 0%;

0%

0

five

0

five

0

ICO-11 (LFA-1 antigen) 50%;

ICOT2 (myelomonocytic antigen)

NAEZ (glycophorin A) 0%;

NAE9 (erythroblast antigen) 0%;

IKO-15 (ligan E-receptor) 72%;

ICO-20 (DM 38-antigen) 0.5%;

T5D (T-cell antigen) 35%;

Type 7 diabetes (T-cell antigen) 25.5%;

SD10 (OAG-ALL-antigen) 76%;

IKO-35 (OAG-ALL-antigen) 17%;

Slg (surface immunoglobulins) 4%.

Immunological diagnosis - T-1-ALL.

The positive effect is that monat ICO-35, obtained using the proposed strain, determines the antigen OAG-ALL on blast cells, the percentage of which can be judged on the immunological variant of leukemia.

The advantage of using mono ICO-35 compared to the prototype (monAT 15) is that monat ICO-35 does not react with granulocytes, which allows more accurate identification of the expression of OAG-ALL on blast cells.

Claims (1)

Invention Formula Strain of hybrid cultured animal cells Mus musculus L. BCKK (II) No. 69 D-producer of monoclonal antibodies against the common antigen of cells of acute lymphoblastic leukemia.