CS244396B1 - Mousy lymphocytoid hybridome img czas mkm-32 - Google Patents

Abstract

The solution concerns a higher lymphocyte hybridoma, producing an antibody against the membrane antigen of human T cells, stored in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation MEM - 32. The monoclonal antibody of the hybridoma MEM-32 itself is suitable for use in enzyme-immunological analysis of human T cells.

Description

(54)

HILGERT IVAN RNDr., CSc.,* HOŘEJŠÍ VÁCLAV,RNDr., CSc.) KRIStOFOVA HANA RNDr., PRAGUE ^*

Mouse lymphocyte hybridoma IMG + CZAS + MEM - 32

The solution concerns a higher lymphocyte hybridoma, producing an antibody against the membrane antigen of human T cells, stored in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation MEM - 32. The monoclonal antibody of the hybridoma MEM-32 itself is suitable for use in enzyme-immunological analysis of human T cells.

244,396

- 1,244,396

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusion of a mouse myeloma line cell Sp2/O-Ag14 and a mouse spleen lymphoid cell, producing an antibody against a membrane antigen present on human T cells (i.e. T lymphocytes). T cells are of fundamental importance in the immune system. The number of T cells and their ratio to B lymphocytes differ from the physiological norm in immunodeficiencies and other pathological conditions.

Up to now, antibodies against membrane antigens have been produced by repeatedly injecting cell suspensions or membrane fractions into production animals, most often rabbits. The serum of animals immunized in this way, collected after a certain period of exposure to the antigen, serves as a source of antibodies. For example, antibodies against thymocytes are prepared in this way, which are used for immunosuppression in organ transplantation in clinical medicine. This procedure, called conventional immunization, has the irremovable disadvantage that the sera contain antibodies against many membrane antigens, some of which are present on more than one type of cell; therefore, the sera cannot be used to finely distinguish cell types and classes.

The disadvantages of conventional sera against membrane antigens are in principle eliminated by the use of hybridoma technology. The product of a hybridoma - a monoclonal antibody - is always directed against a single antigenic determinant. If a sufficiently large set of hybridomas is constructed, it is likely that a hybridoma synthesizing an antibody that recognizes an antigenic determinant characterizing a certain type or class or subclass of lymphocytes will be found.

Until now, the number of T cells has usually been determined by the so-called rosette test with sheep erythrocytes. According to published results (Kung, P.C., Talie, M„A., DeMaria, M.E., Butler, M.S., Lifter, J., Goldstein, G.: Strategies for generating monoclonal anti- 2-. 244 396 bodies defining human T-lymphocyte differentiation antigens. Transplant. Proc. 12, Suppl..1:141-146, 1980) it can be assumed that monoclonal antibodies specifically detecting T lymphocytes can be prepared, which could replace the rosette test currently used.

A significant advance in the diagnosis of human lymphocytes, manifested in a higher reliability of the analytical result, will be achieved if a hybridoma producing a monoclonal antibody against antigens present on human T cells is available, stored in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská no. 1083, under the designation IMG CZAS MEM-32.

The hybridoma was obtained in a manner known from the literature (Fazekas de St. Groth, S., Scheidegger, D.: Production of monoclonal antibodies: Strategies and tactics, J. Immunol. Meth., 35:1-21, 1980; Galfré, G., Howe, S.C., Milstein, C., Butcher, G.W., Howard, J.C.: Antibodies to major histocompatibility antigens produced by hybrid cell lines, Nature, 266:550, 1977) by cloning a set of hybrid cells obtained from the spleen of BALE/c mice immunized with a membrane fraction of human thymus cells.

The advantage of hybridomas is that they produce a homogeneous monoclonal antibody that is capable of specifically reacting with human T cells. The MEM-32 hybridoma can be cultivated in vitro in media suitable for animal cells and is adapted for growth in vivo in the peritoneal cavity of the BALE/c mouse strain. It is possible to start antibody production from preserves stored in liquid nitrogen without additional antigens. The antibody produced by the MEM-32 hybridoma reacts specifically with human T cell populations and there is no need to remove ballast antibodies.

- 3 In order to multiply hybridoma cells in vivo, 4 x 10$ cells were applied to the peritoneal cavity of mice. In order to achieve better attachment of the applied cells, the mouse was treated with paraffin oil (0.5 ml intraperitoneally) 14 days before the transfer of hybridoma cells. After 10 days of hybridoma growth in the peritoneal cavity, the mouse was sacrificed and the unproduced ascitic fluid was collected. A total of 3 ml of ascitic fluid was obtained, which contained^ mg/ml of immunoglobulin. The monoclonal antibody reacted specifically with β T cells of peripheral blood in an indirect enzyme immunoassay. Monocytes, granulocytes and erythrocytes did not react with the antibody. When using purified populations of T and B lymphocytes, the reactivity of the MEM-32 antibody was found with more than 95% of T cells and with less than 5% of B cells. The specificity of the antibody is identical to the commercial monoclonal antibody OKT-1, as demonstrated by repeated parallel determinations including the use of mixtures of both antibodies, both in the case of a mixture of peripheral lymphocytes and in isolated T and B populations (see table).

Tab. Reactivity of antibodies produced by hybridoma MEM-32 and OKT-11 with peripheral blood cells __ _ _ _# ___ ____

Human peripheral blood cells^ % of positive cells reacting with monoclonal antibody in enzyme immunoassay ³ _gmgs

MEM-32 0KT1 MEM-32 + 0KT1

lymphocytes 70-80 70-80 70-80 T lymphocytes >95 >95 >95 B lymphocytes ⁵ <5 <5 <5 monocytes <1 <1 granulocytes <1 <1 <1 erythrocytes <1 customs <1

1Commercial monoclonal antibody from Ortho Pharmaceutical Corporation, Raritan, New Jersey, USA.

*

Human peripheral blood cells were isolated using the described method

244 39Β (Béyum, A. i Isolation of lymphocytes, granulocytes and macrophages. Scand.J.Immunol. 5, Suppl. 5:9, 1976) on a Verografin-Ficoll gradient.

^A slightly modified procedure was used according to the work of Lansdorp, P.M., Astaldi, G.C.B., Oosterhof, F., Janssen, M.C., Zeijlemaker, W.P.: Immunoperoxidase procedures to detect monoclonal antibodies against cell surface antigen. Quantitation of binding and staining of individual cella. J. Immunol.Meth. 39:393-405, 1980.

Lymphocytes were isolated by passing a mixture of lymphocytes through a nylon wool column using the method described (Julius, M.H., Simpson, E., Herzenberg, L.A.: A rapid method for the isolation of functional thymus-derived murine lymphocytes. Europ.J. Immunol. 3:645-649, 1971).

Lymphocytes were isolated by specific adsorption onto plastic plates coated with an antibody against human immunoglobulin according to the method described (Máge, M.G., McHugh, L.L., Rothstein, T.L.: Mouse lymphocytes with and without surface immunoglobulin: Preparative scale separation in polystyrene tissue culture dishes coated with specifically purified anti-immunoglobulin. J.Immunol.Meth. 15:47-56, 1977).

The identity of the antigens recognized by the MEM32 and OKT-1 antibodies was demonstrated by isolating the respective antigens by immunoaffinity chromatography on immobilized MEM-32 antibody and demonstrating strong reactivity of this antigen with OKT-1 antibody. This demonstration was performed as follows: Partially purified MEM-32 antibody was bound to CNBr-Sepharose 4B (Pharmacia, Uppsala, Sweden) (5 mg/ml). The crude membrane fraction from human thymocytes was solubilized in a solution containing 1% NP-40 detergent and the obtained extract was applied to a Sepharose 4B column with covalently bound MEM-32 antibody. After extensive washing, the specifically adsorbed antigen was eluted with 2% sodium dodecyl sulfate solution and the obtained sample was analyzed by SDS-polyacrylamide gel electrophoresis. After electrophoresis, the separated proteins were electrophoretically transferred to a nitrocellulose membrane (Towbin, H., Stachelin, T., Gordon, J.: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc.Natl.Acad.Sci. U.S. 76: 4350-4354, 1979) and detected by indirect enzyme-immunological assay using the appropriate monoclonal antibody and pig anti-mouse antibody conjugated with peroxidase (ÚSQL, Prague). Both the MEM-32 antibody and OKT-1 reacted strongly with the isolated antigen (m.w. 67,000).

MEM-32 hybridoma cells have an ultrastructural picture of typical myeloma cells. The cytoplasm contains a large number of polyribosomes, mitochondria and a poorly developed endoplasmic reticulum. In vitro they grow as semi-suspension cultures. The basic culture medium is Eagle's minimal essential medium with Hanks' salt mixture supplemented with non-essential amino acids, L-glutamine (3mM), sodium pyruvate (1mM). This medium (designated as H-MEMd, Institute of Molecular Genetics, Czech Academy of Sciences) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (0.05 mM), HEPES buffer (10 mM) and inactivated bovine serum (Bioveta, Ivanovice na Hané, 10%). The hybridoma is cultured at 37°C. The median generation time is 21 hours and 5 months after construction the modal chromosome number was 92. The antibody produced is a monoclonal immunoglobulin of the IgGI subclass with kappa light chains, its isoelectric point is pH 6.0 - 6.4.

The monoclonal antibody produced by the MEM-32 hybridoma reacts specifically with human T cells. The MEM-32 hybridoma can be used industrially as a source of monoclonal antibody against T cells in analytical methods. The MEM-32 hybridoma monoclonal antibody can be used for the determination of T cells in peripheral blood and lymphatic organs in clinical diagnostics in medical institutions, especially in various immunological deficiencies and autoimmune diseases and for the classification of leukemias of T cell origin.

Claims (1)

OBJECT OF THE INVENTION The murine lymphocyte hybridoma IMG CZAS MEM-32 producing a monoclonal antibody of IgG1 subclass against human T cell membrane antigen. Printed by Moravian Printing Works,