CS264752B1 - Mouse lymphocytaric hybridom img czasmem-31 - Google Patents

Abstract

Mouse lymphocyte hybridoma, producing membrane antigen antibody human suppressor and cytotoxic T cells deposited in the Ostav hybridoma collection molecular genetics of the Czechoslovak Academy of Sciences under the designation MEM-31. Monoclonal antibody alone hybridoma MEM-31 is suitable for use enzymimmunologic and immunofluorescent human suppressor and cytotoxic analysis T cells.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line Sp2 / 0-Ag14 and a mouse spleen lymphoid cell, producing an antibody against a membrane antigen present on human suppressor and cytotoxic T cells. Functional studies have shown that suppressor and cytotoxic T cells share a common membrane antigen and form a subclass of lymphocytes with important regulatory functions whose representation in peripheral blood and lymphoid tissues differs from the physiological standard in immunodeficiency.

To date, antibodies against membrane antigens have been produced by cell suspensions or membrane fractions being repeatedly injected into production animals, most commonly rabbits.

Serum of animals immunized in this way, collected after a period of antigen treatment, serves as a source of antibodies. Thus, for example, antibodies against thymocytes are prepared which are used for immunosuppression in organ transplantation in clinical medicine. This procedure, called conventional immunization, has the inevitable disadvantage that sera contain antibodies against many membrane antigens, some of which are present on multiple cell types; sera cannot therefore be used to subtly differentiate cell types and classes.

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 that a hybridoma synthesizing an antibody that recognizes an antigenic determinant characterizing a particular lymphocyte type or class or subclass is likely to be found.

According to the published result (Thomas, Y., Sosman, J., Irigoyen, O., Friedman, S. M.,

Kung, P, C., Goldstein, G., Chess, L., Functional analysis of human T cell subsets defined by monoclonal antibodies. I. Collaborative T-T interactions in the immunoregulation of B cell differentiation. J. Immunol., 125: 2401-2408 (1980), it can be concluded that a monoclonal antibody specifically detecting a subclass of suppressor / cytotoxic human T cells suitable for diganostic use can be produced.

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 the antigen present on human suppressor / cytotoxic T cells is deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences. Vídeňská No. 1,083, under the designation IMG CZAS MEM-31.

Said hybridoma was obtained by a method known in 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 line (Nature, 266: 550, 1977) by cloning a collection of hybrid cells derived from the spleen of BALB / c mice immunized with a membrane fraction of human thymus cells.

The advantage of hybridomas is that it produces a homogeneous monoclonal antibody that is capable of specifically reacting with human suppressor / cytotoxic T cells. The MEM-31 hybridoma is capable of culturing in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of BALB / c mice. From preserves stored in liquid nitrogen, antibody production can be started without additional antigen. The antibody produced by the MEM-31 hybridoma reacts specifically with human suppressor / cytotoxic T cell populations and does not need to be rid of ballast antibodies.

Example

To expand the hybridoma cells in vivo, 4x10 ⁵ cells were injected into the peritoneal cavity of mice. To improve attachment of the cells applied, the mouse was treated with paraffin oil (0.5 ml intraperitoneally) 14 days prior to transfer of the hybridoma cells. After 9 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the produced ascites fluid was collected.

A total of 3.5 ml of an ascites fluid containing 5 mg / ml of immunoglobulin was obtained.

The monoclonal antibody reacted specifically in an indirect immunofluorescence or enzyme-immunoassay with peripheral blood suppressor / cytotoxic T cells. Both 35 and 35 positive cells were detected in the enzyme immunoassay up to 1:10 ascitic plasma dilution and in the indirect immunofluorescence test up to 1:10 dilution. The specificity of MEM-31 is identical to the commercial monoclonal antibody OKT-8 as demonstrated by repeated parallel assays, including the use of mixtures of two antibodies (see Table).

Table

Reactivity of antibodies produced by hybridoma MEM-31 and ΟΚΤβ 'with peripheral blood cells

Peripheral% positive cells reactive with monoclonal cells

3 human blood antibody in immunofluorescence assay '

MEM-31 OKT8 MEM-31 + 0KT8 lymphocytes 23 to 33 25 to 34 25 to 35 monocytes 4 1 4 1 4 1 granulocytes 4 1 4 1 41 erythrocytes <1 4 ¹ <1

¹ Commercial monoclonal antibody from Ortho Pharmaceutical Corporation-Raritan,

New Jersey USA.

Human peripheral blood cells were isolated as described (Boyum, A .: Isolation of lymphocytes, granulocytes above macrophages. Scand. J. Immunol. 5, Suppl. 5: 9, 1976) on a Verografin-Ficoll gradient.

An indirect immunofluorescence technique using a fluorescein isothiocyanate-labeled porcine anti-mouse antibody was used.

MEM-31 hybridoma cells have an ultrastructural image of typical rayeloma cells. Cytoplasma contains a large number of polyribosomes, mitochondria and poorly developed endoplasmic reticulum. 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 (3 mM), sodium pyruvate (1 mM). This medium (referred to as H-MEMd) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (0.05 mM), HEPES buffer (10 mM) and inactivated bovine serum (10%) for MEM-31 hybridoma culture. The hybridoma is cultured at 37 ° C. The mean generation time is 18.4 h and 5 months after construction, the modal number of chromosomes was 89. The antibody produced is monoclonal. immunoglobulin of the IgG2a subclass with kappa light chains, its isoelectric point is p1 7.4 to 7.6.

Monoclonal. the antibody produced by the MEM-31 hybridoma reacts specifically with human suppressor / cytotoxic T cells. The MEM-31 hybridoma can be industrially used as a source of monoclonal antibody against suppressor / cytotoxic T cells in analytical methods. The monoclonal antibody of the MEM-31 hybridoma can be used for the determination of suppressor / cytotoxic T cells in peripheral blood and lymphatic organs in clinical diagnosis in healthcare facilities, particularly in the follow-up of healing after organ transplantation, various immunological deficiencies and autoimmune diseases and classification of T leukemia of cell origin.

Claims (1)

Mouse lymphocyte hybridoma IMG CZAS MEM-31 producing a monoclonal antibody immunoglobulin of IgG2a subclass against the membrane antigen of human suppressor and cytotoxic T cells.