CS264752B1 - Lymphocyte cell mouse IMG CZASMEM-31 - Google Patents

Abstract

Mouse lymphocyte hybridoma, producing an antibody against the membrane antigen of human suppressor and cytotoxic T cells, deposited in the hybridoma collection of the Molecular Genetics Collection of the Czech Academy of Sciences under the designation MEM-31. The monoclonal antibody of the MEM-31 hybridoma itself is suitable for use in enzyme-immunological and immunofluorescence analysis of human suppressor and cytotoxic T cells.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusion of a mouse myeloma line cell Sp2/0-Agl4 and a mouse splenic 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, the representation of which in peripheral blood and lymphoid tissues differs from the physiological norm in immunodeficiencies.

So far, 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 thus immunized, 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 unavoidable 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 (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., 125:2 401 to 2 408, 1980) it can be assumed that a monoclonal antibody can be prepared specifically detecting a subclass of suppressor/cytotoxic human T cells suitable for diagnostic use.

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 an antigen present on human suppressor/cytotoxic T cells is available, stored in the Hybridoma Collection of the Molecular Genetics Collection of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská no. 1 083, under the designation IMG CZAS MEM-31.

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 BALB/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 human suppressor/cytotoxic T cells. The MEM-31 hybridoma can be cultured in vitro in media suitable for animal cells and is adapted for growth in vivo in the peritoneal cavity of BALB/c mice. It is possible to start antibody production from preserves stored in liquid nitrogen without additional antigen. The antibody produced by the MEM-31 hybridoma reacts specifically with populations of human suppressor/cytotoxic T cells and there is no need to remove ballast antibodies.

Example

In order to propagate hybridoma cells in vivo, 4xl0 ⁵ cells were injected into the peritoneal cavity of mice. To improve the adhesion of the injected cells, the mice were treated with paraffin oil (0.5 ml intraperitoneally) 14 days before the transfer of hybridoma cells. After 9 days of hybridoma growth in the peritoneal cavity, the mice were sacrificed and the produced ascitic fluid was collected.

A total of 3.5 ml of ascitic fluid was obtained, which contained 5 mg/ml of immunoglobulin.

The monoclonal antibody reacted specifically in an indirect immunofluorescence or enzyme immunoassay with peripheral blood suppressor/cytotoxic T cells. Both methods detected 25 to 35 positive cells in the enzyme immunoassay up to a 1:10 dilution of ascitic plasma and in the indirect immunofluorescence assay up to a 1:10 dilution. The specificity of MEM-31 is identical to the commercial monoclonal antibody OKT-8, as demonstrated by repeated parallel determinations including the use of mixtures of the two antibodies (see table).

Table

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

Peripheral cells % of positive cells reacting with monoclonal

3 human blood antibody in immunofluorescence test'

MEM-31 OCT8 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 by the described method (Boyum, A.: Isolation of lymphocytes, granulocytes and macrophages. Scand. J. Immunol. 5, Suppl. 5:9, 1976) on a Verografin-Ficoll gradient.

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

The MEM-31 hybridoma cells have the ultrastructural appearance of typical rayeloma 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 (3 mM), sodium pyruvate (1 mM). This medium (designated as H-MEMd) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (0.05 mM), HEPES buffer (10 mM) and inactivated bovine serum (10%) for the cultivation of the MEM-31 hybridoma. The hybridoma is cultured at 37 °C. The median generation time is 18.4 h and 5 months after construction the modal chromosome number was 89. The antibody produced is monoclonal. an immunoglobulin of the IgG2a subclass with kappa-type light chains, its isoelectric point is pI 7.4 to 7.6.

Monoclonal antibody produced by hybridoma MEM-31 reacts specifically with human suppressor/cytotoxic T cells. Hybridoma MEM-31 can be used industrially as a source of monoclonal antibody against suppressor/cytotoxic T cells in analytical methods. Monoclonal antibody of hybridoma MEM-31 can be used for determination of suppressor/cytotoxic T cells in peripheral blood and lymphatic organs in clinical diagnostics in medical institutions, especially in monitoring rejection crises after organ transplantation, in various immunological deficiencies and autoimmune diseases and for classification of leukemia of T 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.