CS275658B6 - Mouse Lymphocyte Hybrid Bra FB6 / a - Google Patents

Abstract

The solution concerns the mouse lymphocyte hybridoma Bra FB6/a, producing a monoclonal antibody of the IgG2b subclass against the HLA antigen class II (DP subtype), which 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. The monoclonal antibody Bra FB6/a is suitable for immunophenotyping of some tumors, refining the immunocytochemical diagnosis of some types of neoplasms, monitoring the presence of DP antigen on the cells of some neoplasms and normal immunocompetent cells, experimental and diagnostic procedures in transplantation immunology and biochemical (immunochemical) isolation of DP antigen.

Description

The invention relates to a murine Bra FB6 / lymphocyte hybridoma, i.e. a hybrid culture of cells of a single cell (monoclonal) origin, obtained by cell fusion of a mouse myeloma cell line Sp2 / 0 and spleen lymphoid cells of syngeneic experimental animals immunized with human non-T, non-B ALL leukemia cell line. line. The hybridoma produces both in vitro and in vivo murine IgG _2b monoclonal antibody recognizing human HLA antigen II. class (DP).

Floods, antibodies to cell surface antigens were obtained by conventional immunization of experimental animals, resulting in a heterogeneous mixture of antibodies of cut classes and subclasses as well as immunochemical specificities. As a rule, cross-reactivities of such preparations cannot be eliminated by in vitro saturation, which is a non-standard procedure requiring large numbers of saturating cells.

Said disadvantages with regard to antibodies against HLA antigen II. class, removes the solution according to the invention, which consists in obtaining a murine lymphocyte hybridoma Bra FB6 / a, producing a monoclonal antibody of class IgG _2b , against HLA antigen II. DP subtype class. This hybridoma according to the invention 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. Csl. armády 21, 812 32 Bratislava, under the designation Bra FB6 / 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; a Bra FB6 / a subclone with high antibody production was obtained by repeated limiting dilution dilution.

The advantage of the obtained Bra FB6 / a hybridoma according to the invention is in particular that it produces monoclonal antibodies recognizing the HLA antigen of class II DP and specifically immunoprecipitates the glycopeptide chains of gp (29,34), which has experimental use in laboratories for transplant immunology and oncology. .

An example of spleen cells from an immunized BALB / c inbred mouse is fused with 6.10 Sp2 / 0 myeloma cells in the presence of 45% polyethylene glycol (PEG), cloned by limiting dilution on a syngeneic mouse thymocyte layer (2.10 ⁵ cells / microculture) in hypoxinant selection medium. aminopterin-thymidine (HAT).

The Bra FB6 / subclone, with a high production of monoclonal antibodies selected on the basis of specific reactivity with a panel of human leukemia / lymphoma cell lines and hematopoietic cells from healthy donors, was obtained by repeated cloning by limiting dilution technique in microplates in 10% fetal body culture medium. until a stable monoclonal cell culture producing a monoclonal antibody of a given Specificity is formed.

Production of the monoclonal antibody in a high concentration is carried out such that i

CS 275758 B6 inoculates 5.10 2 hybridoma cells into the peritoneal cavity of syngeneic mice,

Treated by intraperitoneal administration of tetramethylpentadecane. Monoclonal antibody, § present in the cell culture supernatant resp. in ascitic fluid obtained from inoculated mice, specifically recognizes HLA antigen II. class of the DP subtype and Specifically immunoprecipitates the glycopeptide chains of gp (29,34).

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Growth hybridoma cells as a suspension culture of cells with the morphology of mouse myeloma cells. The basic culture medium is RPMI 1640 with 10% fetal calf serum.i ·

The hybridoma is cultured at 37 ° C in 0.5% carbon dioxide, from frozen ampoules re-culturing of the hybridoma with the production of a monoclonal antibody with the original specificity is possible to test samples of the flood hybridoma (in liquid nitrogen).

Mouse lymphocyte hybridoma Bra FB6 / and monoclonal antibody produced with it they can be technologically prepared and used commercially in the diagnostic immunophenotyping of tumor cells, in the immunocytochemical advanced diagnostics of some types;

pov tumors, in evaluating the metastatic potential of cells of some neoplasms, i;

in the Study of the Induction of Membrane Antigen Expression by Lymphokines and Immunomodulators, in Transplant Immunology Techniques, in the Biochemical Isolation of HLA Antigen (DP) II. Classes

Claims (1)

4.105 of hybridoma cells are inoculated into the peritoneal cavity of syngeneic mice, determined by intraperitoneal administration of tetramethylpentadecane. The monoclonal antibody present in the cell culture supernatant, respectively. in ascitic fluid obtained from inoculated mice, Specifically recognizes HLA antigen IX. of the DP subtype and specifically immunoprecipitates the gp (29,34) glycopeptide chains. Growth hybridoma cells as a suspension cell culture with mouse myeloma cell morphology. The basic culture medium is RPMI 1640 with 10% fetal calf serum. The hybridoma is cultured at 37 ° C in 0.5% carbon dioxide, a new culture of the monoclonal antibody-producing hybridoma is possible from frozen, pooled hybridoma samples (in liquid nitrogen) with Specific Specificity. The murine lymphocyte hybridoma Bra FB6 / and the monoclonal antibody produced by it can be technologically prepared and commercially used in diagnostic immunophenothesis of tumor cells, in immunocytochemical refined diagnosis of some tumor types, in the evaluation of metastatic potential of cells of some neoplasms, in the study of induction of lymphokine membrane antigen expression and immunomodulators, in transplant immunology techniques, in the biochemical isolation of HLA antigen (DP) class II. BACKGROUND OF THE INVENTION The murine lymphocyte hybridoma Bra FB6 / a, producing a transdermal antibody against the HLA antigen II subclass. of the DP subtype. $ .1 • IT <? • t