CS275657B6 - MURINE LYMPHOCYTE HYBRIDOMA Bra 23/9/a - Google Patents

Abstract

The solution concerns the mouse lymphocytic hybridoma Bra 23/9/a producing a monoclonal antibody of the IgG subclass against the HLA class 1 antigen - non-poly-3 morphic determinant, which is stored in the collection of hybridomas and cell cultures of the Department of Molecular Immunology, Institute of Experimental Oncology SAS, ul. Csl. armády 21, 812 32 Bratislava. The monoclonal antibody Bra 23/9/a is suitable for immunophenotyping of tumor cells, for refining immunocytochemical diagnostics of some tumors, monitoring the expression of transplantation antigens on normal and tumor cells, in the laboratory technique of transplantation immunology, in the biochemical isolation of HLA class 1 antigens.

Description

The invention relates to a mouse lymphocytic hybridoma Bra Z ~ 519 / a ie a hybrid culture of cells of a single cell (monoclonal) obtained, obtained by cell fusion of the mouse myeloma cell line Sp2 / 0 and spleen lymphoid cells of syngeneic experimental animals immunized with human non-T, non-B ALL leukemic cell line. The hybridoma produces both in vitro and in vivo murine IgG _2a class monoclonal antibody, recognizing human HLA class I antigen (a non-polymorphic determinant).

Floods, antibodies against cell surface natigens were obtained by conventional immunization of experimental animals, resulting in a heterogeneous mixture of antibodies of different classes and subclasses as well as immunochemical specificities. In general, cross-reactivities of such preparations cannot be eliminated by in vitro saturation, which is also a non-standard procedure requiring significant amounts of saturating cells.

The above-mentioned disadvantages with regard to antibodies against HLA antigens (MHC class I complex antigens) are eliminated by the solution according to the invention, which consists in obtaining a mouse lymphocyte hybridoma producing an IgG _2a class monoclonal antibody against HLA class I antigen ( non-polymorphic determinant). This hybridoma is stored in the collection of hybridomas and cell cultures of the Department of Molecular Immunology of the Institute of Experimental Oncology of the Slovak Academy of Sciences, ul. armády 21, 812 32 Bratislava, under the designation Bra 23 / / 9 / a.

Said hybridoma was obtained by a method known from the literature (Kohler, G., Milstein, C .: Nature 256 (1975), 495), by cloning cells obtained by somatic hybridization of the mouse myeloma cell line Sp2 / 0 and spleen lymphoid cells of a syngeneic animal (inbred breeding BALB / c), immunized with repeated intraperitoneal doses (2.10 cells / dose) of a cell suspension of a human non-T, non-B ALL leukemia cell line; subclone Bra 23/9 / and with high antibody production was obtained by repeated cloning limiting dilution.

The advantage of the unique hybridoma obtained according to the invention lies in particular in the production! monoclonal antibody recognizing human HLA class I antigen and specifically immunoprecipitating the glycopeptide chain gp 44, p 12, C0, can be used experimentally in the laboratory in transplantation immunology and oncology.

Example

7 spleen cells of immunized BALB / c inbred mice are fused with 6.10 'Sp2 / 0 myeloma cells in the presence of 45% polyethylene glycol (PEG), cloned by limiting dilution on a layer of syngeneic mouse thymocytes (2.10 ⁵ cells / microculture) in selection medium. aminopterin-thymidine (HAT).

Subclone Bra 23/9 / and with a high production of monoclonal antibodies, selected on the basis of specific reactivity with a panel of human leukemia / lymph cell lines and hematopoietic cells from healthy donors, was obtained by repeated cloning by limiting dilution technique in microplates in 10% fetal culture medium. calf serum, until a stable monoclonal cell culture is produced, producing a monoclonal antibody of a given specificity.

Production of a monoclonal antibody of a given specificity at a high concentration is performed by inoculating 5.10 ⁶ hybridoma cells into the peritoneal cavity of syngeneic mice, premedicated by intraperitoneal administration of tetramethylpentadecane. Mono

CS 275657 B6 2 clonal antibody, present in the cell culture supernatant or in the ascites fluid obtained from inoculated mice, specifically recognizes human HLA class I antigen (non-polymorphic determinant) and specifically immunoprecipitates glycopeptide chains j gp ( pl2). í

Growth hybridoma cells as a suspension culture of cells with morphological mouse myeloma cells. The basic culture medium is RPMI 1640 with 10% fetal calf serum. ) The hybridoma is cultured at 37 ° C in 0.5% pxide carbon dioxide. Of frozen ampoules; samples of the original hybridoma (in liquid nitrogen), it is possible to recultivate the hybridoma to produce a monoclonal antibody with the original specificity. i

Mouse lymphocyte hybridoma Bra 23/9 / and the monoclonal antibody produced by it can be technologically prepared and commercially used in diagnostic phenotyping of tumor cells, in diagnostics by immunocytochemical methods of some types of neoplasms, in evaluation of metastatic potential of tumor cells, in In vitro study * induced expression of membrane antigens, in immunochemical purification of HLA antigens I. treida. ;

Claims (1)

PATENT CLAIMS Mouse lymphocyte hybridoma Bra 23/9 / a, producing a monoclonal antibody of the IgG2 subclass _and against the human HLA class I antigen - a non-polymorphic determinant.