CS253687B1 - Mouse lymphocyte hybridoma IMG CZA3 MEM-57 - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody against the membrane antigen of human,? cells, stored in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation MEM-57. The monoclonal antibody of the hybridoma MEM-57 itself is suitable for use in enzyme-immunological analysis or indirect immunofluorescence analysis of the T3 antigen of T lymphocytes.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line 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 differs from physiological norm in immunodeficiency and other pathological conditions.

Antibodies against antigens present on a particular cell subpopulation cannot be prepared by direct immunization of animals with a lymphocyte cell suspension or membrane fraction, i.e., conventional immunization. Conventional immunization has the unavoidable disadvantage that sera contain antibodies against many membrane antigens, some of which are present on multiple cell types; therefore, sera cannot be used to subtly differentiate cell types and classes. Preparation of pure antigen without contamination with other membrane components for immunization is laborious and mostly unsuccessful.

In principle, the use of hybridoma technology eliminates the disadvantages of conventional sera against membrane antigens. The hybridoma product - a monoclonal antibody - is directed against a single antigenic determinant. If a sufficiently large set of hybridomas is constructed, it is likely to be able to identify an antibody synthesizing hybridoma that recognizes an antigenic determinant characterizing a particular type or class or subclass of lymphocytes.

There are a number of antigens on T lymphocytes that differ in molecular weight, chemical structure and physiological function.

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For detailed analysis of antigens on T lymphocytes, it is essential to have antibodies against as many membrane antigens as possible. According to published results / Kúng, P.G., Talie, M.A., DeMaria, M.E., Butler, M.S., Lifter, J., Goldstein, G .: Strategies for Generating Monoclonal Antibodies Defining Human T-lymphocyte Differentiation Antigens. Transplant. Why. 12, Suppl. 1: 141-146, 1930], it can be concluded that monoclonal antibodies specifically detecting various T cell antigens can be prepared.

Substantial progress in the diagnosis of human lymphocytes, which results in higher reliability of the analytical result, will be achieved if a hybridoma producing a monoclonal antibody against T3 antigen present on human T cells is available, deposited in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences. 1083, under the designation IMG GZAS MEM-57.

Said hybridoma was obtained by a method known in the literature. 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 / cloning a set of hybrid cells derived from the spleen of BALB / c mice immunized with a membrane fraction of human thymus cells.

The advantage of the hybridoma is that it produces a homogeneous monoclonal antibody that is capable of specifically reacting with human T cells. The MEM-57 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of BALB / c mouse mice. From preserves stored in liquid nitrogen, antibody production can be started without additional antigen. The antibody produced by the MEM-57 hybridoma reacts specifically with human T cell populations and does not need to be rid of ballast antibodies.

Example

In order to propagate hybridoma cells in vivo, apli253 687 was applied

- 5 forged 3 x 10 ⁶ cells into the peritoneal cavity of mice. To improve attachment of the cells applied, the mice were treated with paraffin oil (0.5 ml intraperitoneally) 14 days prior to transfer of the hybridoma cells. After 10 days of hybridoma growth in the peritoneal cavity, mice were exterminated and unproduced ascites fluid was collected. A total of 3 ml of ascites fluid was obtained, which contained 5 µg / ml of immunoglohulin. The monoclonal antibody reacted specifically with peripheral blood T cells in an indirect enzyme immunoassay. Monocytes, granulocytes and erythrocytes did not react with the antibody. Using purified populations of T and B cells, the reactivity of the MEM-57 antibody was found to be greater than 95% T cells and less than 5% B cells. The specificity of the antibody is identical to the commercial monoclonal antibody OKT-3, as demonstrated by repeated parallel assays, including the use of mixtures of both antibodies, both for a mixture of peripheral lymphocytes and isolated T and B populations (see Table).

Tab. Reactivity of antibodies produced by the hybridoma MEM-57 1 ^v and QKT-3 with peripheral blood cells

Peripheral human blood cells2% positive antibody-reactive cells in the enzyme-immunoassay OMM-57 assay

0KT3

MEM-57 + 0KT3 lymphocytes 70-80

T lymphocytes ^ 95

B lymphocytes5 monocytes 1 granulocytes 1 erythrocytes 1

70-80

70-80

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

Human peripheral blood cells were isolated by the method of B. Yum, A., Isolation of lymphocytes, granulocytes and macrophages. Scand. J. Immunol. 5, Suppl. 5: 9, 1976) on a Verografin-Eicoll gradient.

Lansdorp, P. M., Astaldi, G.O.B., Oosterhof, F., Janssen, M. C., Zeijlemaker, W.P .: Immunoperation procedures to detect monoclonal antibodies against cells

253 687 surface antigen. Quantitation of binding and staining of individual cells. J. Immunol. Meth. 59: 595-405 (1980).

lymphocytes were isolated by passing the mixture of lymphocytes through a nylon cotton column as described in (Julius, M.H., Simpson, E., Herzenberg, L.A .: A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol. 5: 145-649 (1971)].

lymphocytes were isolated by adsorption to plastic plates coated with an anti-human immunoglobulin antibody as described / Mage, MG, McHugh, LL, Rothstein, TL: Mouse lymphocytes with and without surface immunoglobulin: preparative scale separation in polystyrene tissue culture dishes coated with targeted anti-immunoglobulin . J. Immunol. Meth. 15: 47-56, 1977].

Three approaches were used to demonstrate that MEM-57 recognizes T5 antigen on the surface of T lymphocytes; a) The monoclonal antibody MEM-57 precipitated from surface iodinated cells (Morrison, M .: Lactoperixidase-catalyzed iodination as a tool for investigation of protein). Methods Enzymol. 70: 214-220, 1980 (T line HPB-AL) antigen fraction of mol. 25 kDa mass, demonstrated by polyacrylamide gel electrophoretic analysis in the presence of sodium dodecyl sulfate; mol. the weight of this antigen fraction corresponds to mol. weight of T5 antigen.

b) MEM-57 antibody at a concentration of 10-20 ng / ml had mitogenic properties demonstrated by induction of DNA synthesis (via thymidine incorporation) in human peripheral blood lymphocytes. The same mitogenic effects have been reported for the 0KT5 antibody / Van Wauwe, J.P., DeMey, J.R., Goossens, J.G .: 0KT5: A monoclonal anti-human T lymphocyte antibody with potent mitogenic properties. J. Immunol. 124: 2708-2715, 1980].

c / The identity of the antigens recognized by the MEM-57 and 0KT5 antibodies was demonstrated by first moving the antigen reactive with MEM-57 to one pole of T cells, called capping followed by uptake of the antigen into most cells, and then reactivity of these altered cells tested with antibody 0KT5 ·

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- 5 that after T3 antigen removal (MEM-57 antibody) the ability of T lymphocytes to bind OKT3 antibody also disappears, whereas reactivity with antibody of unrelated specificity B2M-01 / Hilgert, I., Horejsi, V., Krestofova, H .: The use of murine monoclonal antibody B2M-01 for detection and 'purification of human 2 “ ^m i ^cr ° ·· globulin. Pole. biol. (Prague) (30: 569-376, 1984) will not change. This assay was performed by incubating T lymphocytes for 30 min at 20 ° C in MEM-57 antibody solution and then for 30 min at 37 ° C with porcine anti-mouse immunoglobulin antibody. The cells thus prepared were subjected to a conventional indirect immunofluorescence assay, i.e. incubation with MEM-57, 0KT3,

B2M-01 and in a second step with a fluorescein isothiocyanate labeled porcine anti-mouse immunoglobulin. T cells were washed with saline before incubation with the following antibody.

MEM-57 hybridoma cells have an ultrastructural pattern of typical myeloma cells. They grow in vitro as semi-suspension cultures. The basic culture medium is Eagle's minimum essential medium with Hanks salt mixture supplemented with non-essential amino acids, L-glutamine (3mM), sodium pyruvate (IraM). This medium (referred to as RPMI, Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (0.05 mM), HEPES buffer (10 mM) and inactivated bovine serum for cultivation of MEM-57 hybridoma (Bioveta, Ivanovice). in Haná, 10% /. The hybridoma is cultured at 37 ° C. The mean generation time is 18 hours and 5 months after construction, the modal number of chromosomes was 88. The antibody produced is a monoclonal immunoglobulin of the IgG2a subclass with light chains of kappa type, its isoelectric point is pH 7.4 - 8.0.

The monoclonal antibody produced by the hybridoma MEM-57 reacts specifically with human T cells. The MEM-57 hybridoma can be industrially used as a source of monoclonal antibody against T cells in analytical methods. The monoclonal antibody of the hybridoma MEM-57 can be used for the determination of T cells in peripheral blood and lymphatic organs in clinical diagnosis in medical facilities, particularly in various immuno253 687

- 6 logical deficiencies and autoimmune diseases and the classification of T cell leukemias. The monoclonal antibody can also be used in therapy as an immunosuppressant in healing crises in clinical transplantation or to specifically remove T lymphocytes in bone marrow transplantation.

Claims (1)

SUBJECT OF THE INVENTION IMG CZAS MEM-57 mouse lymphocyte hybridoma producing a monoclonal antibody of IgG2a subclass against human T cell membrane antigen T3.