CS240807B1 - Mouse lymphatic hybridom imb czas tec-%1 - Google Patents

Abstract

the solution relates to mouse lymphocyte hybridoma. antibody-producing antibody human granulocyte membrane antigen deposited in the Institute's hybridoma collection molecular genetics of the Czechoslovak Academy of Sciences under the designation TEC-01. Hybridoma monoclonal antibody TEC-01 is suitable for the identification of granulocytes human peripheral blood and myeloid leukemic cells, their elimination from cell suspensions and target antigen analysis structure.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line P3-X63-Ag8.653 and a mouse spleen cell producing an antibody against the membrane antigen of human granulocytes.

To date, antibodies against membrane antigens of human granulocytes have been produced by granulocytes isolated from human blood being repeatedly injected as antigen to human volunteers or experimental animals (most commonly mouse, rat, rabbit). Serum of such immunized humans or animals, taken after some time of antigen treatment, serves as a source of antibodies used mainly for the identification and typing of peripheral blood granulocytes. This procedure, called conventional immunization, has a number of drawbacks.

For each immunization and testing of serum antibody activity, it is necessary to isolate granulocytes from peripheral blood.

There is a heterogeneous mixture of antibodies in the sera of both immunized humans and animals, whose spectrum is different and unrepeatable in each organism. The organism generates antibodies against a variety of membrane antigens, and the specificity of the sera depends on how they are absorbed and tested. Production batches of sera prepared by conventional immunization are therefore difficult to standardize and are based on production over a wide quality range.

These drawbacks of the above-mentioned and previously used procedure will be eliminated if a hybridoma producing an antibody against the membrane antigen of human granulocytes, deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská 1083, is available IMG CZAS TEC-01.

The hybridoma was obtained by a method known in the literature (Kohler, G., Milstein, C .: Gontinuous cultures of fused cells secreting antibody of predefined specificity, Nature, 256: 495-497, 1975; Kennett, RH, McKearn, TJ, Bechtol , KB (eds.): Monoclonal antibodies, Hybridomas:

240 807 a new dimension in biological analysis, Plenum Press,

New York and London, 1980). P3-X63-Ag8.653 mouse myeloma line cells and cells obtained from the spleens of BALE / c mice immunized with F9 teratocarcinoma line cells were used for fusion.

The lymphocyte hybridoma TEC-01 produces a homogeneous antibody, the so-called monoclonal antibody, which reacts with human granulocytes and does not react with other types of human peripheral blood cells (monocytes, lymphocytes, erythrocytes). The TEC-01 hybridoma can be cultured in vitro in mammalian cell media and is adapted for growth in the peritoneal cavity of BALE / c mice. From preserves stored in liquid nitrogen, antibody production can be initiated without the need for immunization and serum absorption. The monoclonal antibody produced by the TEC-01 hybridoma is specific for the membrane antigen of human granulocytes and is free of any ballast antibodies.

Example:

To expand TEC-01 hybridoma cells in vivo, 10 'cells were injected into the peritoneal cavity of BALE / c mice. To increase production of ascites fluid, mice were treated with 2,6,10,14-tetramethylpentadecan oil (0.5 ml intraperitoneally) 14 days prior to hybridoma cell administration. After 14 days of hybridoma growth in the peritoneal cavity, the mouse was killed and unproduced ascites fluid was collected. A total of 4 ml of ascites fluid containing 12 mg / ml IgM-class immunoglobulin was obtained.

The monoclonal antibody reacted in an indirect radioimmunoassay with peripheral blood granulocytes up to a 1:10 dilution of ascites fluid and did not react with lymphocytes even at a 1:10 dilution. The results of a direct radioimmunoassay and an indirect immunofluorescence assay showed that from human peripheral blood cells only granulocytes carry the antigen recognized by said antibody (see table).

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Tab. Reactivity of antibodies produced by TEC-01 hybridoma with peripheral blood cells

Cells1

Test used to detect reactivity

3 radioimmunological immunofluorescence (imp. Min. ^)% Shining cells

Lymphocytes, monocytes 300 <2

Granulocytes 12 400 98

Erythrocytes 100 <0.1 *

Human peripheral blood cells were divided according to density on a Percoll gradient

125 A direct radioimmunoassay was used in which the? 1 labeled monoclonal antibody (50,000 imp. Min. ^) Was incubated for 1 hour with 0.25 x 10 ^ cells and cell-bound radioactivity was measured.

An indirect immunofluorescence assay was used in which cells were incubated with 0.1 mg monoclonal antibody; unbound antibody was washed off and cells incubated with FlTC-labeled porcine anti-mouse IgM. After removal of unbound antibody, the cells were examined in a fluorescence microscope.

When tested on human myeloid and lymphoid leukemic cell lines, the TEC-01 antibody bound only to myeloid leukemic cells.

TEC-01 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free and membrane-bound polyribosomes. 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) and sodium pyruvate (1mM).

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This medium (referred to as H-MEMd, Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin and temperature inactivated (56 ° C, 30 min) bovine serum (Bioveta, Ivanovice na Hané, 10%) for TEC-01 hybridoma culture. The hybridoma is cultured at 37 ° C. The mean generation time is 14.6 h; at 6 months after construction, the modal number of chromosomes was 76. The monoclonal antibody produced is an IgM, kappa class immunoglobulin.

The monoclonal antibody produced by the murine lymphocyte hybridoma TEC-01 can be used in healthcare to safely identify peripheral blood granulocytes, to distinguish myeloid leukemia cells from lymphoid leukemia cells, to eliminate myeloid leukemia cells from cell suspensions in vitro, and to biochemical and biological analysis of target antigen .

Claims (1)

SUBJECT OF THE INVENTION Mouse lymphocyte hybridoma IMG CZAS TEC-01, producing an IgM class monoclonal antibody against the membrane antigen of human granulocytes.