CS244200B1 - Mouse lymphocytic hybridoma IMG CZAS MEM-12 - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody against the membrane antigen of human HLA-DR positive cells, stored in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation MEM-12. The monoclonal antibody of the MEM-12 hybridoma itself is suitable for use in enzyme-immunological and immunofluorescence analysis of human HLA-DR positive cells.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusion of a cell of the mouse myeloma line Sp2/O-Ag14 and a mouse splenic lymphoid cell, producing an antibody against the membrane antigen HLA-DR present on human B cells, monocytes, macrophages and activated T cells. These antigens are of fundamental importance for the regulation of various functions of the immune system. Quantitative determination of HLA-DR positive cells is of importance in the diagnosis of some 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 it will be possible to find a hybridoma synthesizing an antibody that recognizes an antigenic determinant characterizing a certain type or class or subclass of lymphocytes.

According to the published result (Brodsky, F.I.I., Parham, P., Bodmer, W.F.: Monoclonal antibodies to HLA-DRw determinant, Tissue Antigens 16:30, 1930) it can be concluded that it is possible to prepare

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-λmonoclonal antibodies specifically detecting monomorphic determinants on HLA-DR antigens, advantageous for diagnostic use. Monomorphic are those determinants that are present in all forms of the HLA-DR antigen in the human population.

A significant advance in the diagnosis of human lymphocytes, manifested by a higher reliability of the analytical result, will be achieved if a hybridoma producing a monoclonal antibody against the monomorphic determinant of human HLA-DR antigens is available, deposited in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňské £· 1083, under the designation IMG CZAS MEM-12.

The hybridoma was obtained by a method 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 BALQ/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 able to react specifically with cells carrying DR antigens. The KEM-12 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 antigen. The antibody produced by the MEM-12 hybridoma reacts specifically with populations of human cells expressing DR antigens and there is no need to remove ballast antibodies.

Example

In order to propagate hybridoma cells in vivo, 200 g of apli244 was used.

4 x 10 cells were injected into 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 11 days of hybridoma growth in the peritoneal cavity, the mouse was sacrificed and the unproduced ascitic fluid was collected. A total of 2.5 ml of ascitic fluid was obtained, which contained 5 mg/ml of immunoglobulin. The monoclonal antibody reacted specifically in an indirect immunofluorescence or enzyme-linked immunosorbent assay with a fraction of peripheral blood lymphocytes. Both methods revealed 25% to 30% positive cells in the enzyme immunoassay up to a dilution of ascitic plasma of 1:10^ and in the indirect immunofluorescence assay up to a dilution of 1:10\ When using purified populations of T and B lymphocytes, the reactivity of the MEM-12 antibody was found with more than 95% of B lymphocytes and 5-10% of T lymphocytes. More than 95% of monocytes were also strongly positive, granulocytes and erythrocytes were negative. The specificity of the MEM-12 antibody is identical to the RF-KLA-DR antibody, as demonstrated by repeated parallel determinations including the use of antibody mixtures, both in the case of a mixture of peripheral lymphocytes and in isolated cell populations (see table).

Tab. Reactivity of antibodies produced by hybridoma MEK-12 and 1 *

RF-HLA-DR with peripheral blood cells

Peripheral cells % of positive cells reacting with monohuman blood clonal antibody in enzyme immunoassay or indirect immunofluorescence 4 test

KEM-12 RF-HLA-DR MEM-12+RP-: lymphocytes 25-30 25-30 25-30 T lymphocytes^ 5-10 5-10 5-10 B lymphocytes^ >95 >95 >95 monocytes >95 >95 >95 granulocytes <1 < 1 < 1 erythrocytes <1 1 <1

- ϊ244 200 ¹ Monoclonal antibody RF-HLA-DR was obtained from Prof. G.

Janossy of The Royal Free Hospital, London.

² Human peripheral blood cells were isolated by the described method (Béyum, A.: Isolation of lymphocytes, granulocytes and macrophages. Scand.J.Immunol. 5, Suppl. 5:9, 1976) on a VerografinFicoll gradient.

^The procedure was used according to 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 cells. J. Immunol. Meth. 39:393-405, 1980.

^Indirect immunofluorescence technique was used using swine anti-mouse immunoglobulin labeled with fluorescein isothiocyanate (ÚSOL, Prague).

^T lymphocytes were isolated by passing the lymphocyte mixture 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 thymue-derived murine lymphocytes. Eur.J.Immunol. 3:645-649, 1971).

Lymphocytes were isolated by adsorption onto plastic plates coated with an antibody against human immunoglobulin according to the method described (Máge, M.G., MoHugh, L.L., Rothstein, T.L.:

Fly 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 MEM-12 and RF-HLA-DR antibodies was demonstrated by isolating the respective antigen by immunoaffinity chromatography on immobilized MEM-12 antibody and demonstrating the reactivity of this antigen with RF-HLA-DR antibody. The antigen obtained by immunoaffinity chromatography on Sepharose 4B with covalently bound MEM-12 antibody provided, after electrophoresis in a polyacrylamide gel in the presence of sodium dodecyl sulfate, a bu3 1-zone corresponding to a m.w. dM65 000

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- £ (sample not heated), or 2 zones corresponding to m.h. 29,000 and 33,000 (sample preheated to 100°C for 2 min), which is a result typical for DR antigens (Shackleford, D.A., Kaufman, J.F., Korman, A.J., Strominger, J.L.; HLA-DR antigene:

Structure, separation of subpopulation, gene cloning and function. Immunol.Rev. 66:133-187, 1982). After electrophoretic transfer of the separated proteins to a nitrocellulose membrane and specific detection by indirect enzyme immunoassay (using the appropriate monoclonal antibody and pig antimouse immunoglobulin conjugated with β peroxidase (ÚSOL, Prague)). Both antibodies (MEM-12 and KF-HLA-DR) reacted strongly with the antigen in dimeric form (m.w. $0<&65 000), the RF-HLA-DR antibody reacted with a subunit with a m.w. 29 000 of the isolated antigen.

MEM-12 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-suspended cultures. The basic culture medium is Eagle's minimal essential medium 8 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, Czechoslovak 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 mean generation time is 16.8 hours and 5 months after construction the modal number of chromoaromatics was 96. The produced antibody is a monoclonal immunoglobulin of the IgGI subclass with kappa-type light chains, its isoelectric point is pH 6.8 - 7.0.

The monoclonal antibody produced by the MEM-12 hybridoma reacts specifically with human B cells, monocytes, macrophages and activated T cells. The MEM-12 hybridoma can be used industrially as a source of monoclonal antibody against DR positive cells in analytical methods. The MEM-12 hybridoma monoclonal antibody can be used for the determination of DR positive 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 and malignant lymphomas.

Claims (1)

SUBJECT OF THE INVENTION Mouse lymphocyte hybridoma IMG CZAS MEM-12 producing a monoclonal antibody of IgG1 subclass against the membrane antigen of human HLA-DR positive cells. Printed by Moravian printing works, center. 11 100, the class of People's Militia 3, Olomouc Price: 2,40 Kčs