CS244399B1 - Mouse lymphocyte hybridoma IMG-CSAZ HLA-01 - Google Patents

Abstract

The present invention relates to a mouse hybridoma producing an antibody against the membrane antigen of human HLA-Bw4 positive cells, stored in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation HLA-01. The monoclonal antibody of the hybridoma HLA-01 is suitable for use in a microcytotoxicity test in the presence of complement and in a direct radioimmunoanalytical binding test for the detection of human HLA-Bw4 positive cells.

Description

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Mouse lymphocyte hybrid IMG-CSAZ hybrid HLA-01

The ee solution relates to a murine hybridoma producing an antibody against the membrane antigen of human HLA-Bw4 positive cells deposited in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the name HLA-01. The HLA-01 hybridoma monoclonal antibody is suitable for use in a microcytotoxic assay in the presence of complement and in a direct radloimmunoassay binding assay for detecting human HLA-Bw4 positive cells.

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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 HLA-Bw4 membrane antigen present on human lymphocytes. HLA antigens are essential for regulating various functions of the immune system. The identification of these antigens is important in the transplantation of vital tissues and organs and in the diagnosis of some pathological conditions.

To date, antibodies to the main human histocompatibility system (HLA) antigens are obtained from the sera of women after repeated pregnancies or from patients after repeated blood transfusions. Although these sera are essential in the definition of HLA antigens, their limitation is that the antisera contain low titer antibodies, are multispecific and cross-reactive. Antisera containing antibodies that identify some rare HLA specificities are difficult to find. For this reason, approaches have been sought that would make it easier to identify HLA antigens. One approach is to immunize volunteers or experimental animals. The serum of humans or animals so immunized after a period of antigen exposure serves as a source of antibodies used to identify HLA antigens. This procedure, called conventional immunization, has a number of drawbacks. Obtaining antibodies from the sera of immunized volunteers from an ethical point of view has no practical use. In the serum of immunized animals there is a heterogeneous mixture of antibodies, the spectrum of which 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. Therefore, antisera prepared in a conventional manner are very difficult to standardize.

The disadvantages of conventional sera against the antigens of the main human histocompatibility system HLA in principle eliminate the use of hybridoma technology. The hybridoma product - a monoclonal antibody - is directed against a single antigenic determinant. If a sufficiently large sorbate of hybridomas is constructed, it is

- x 244 399 is likely to find an antibody synthesizing hybridoma that recognizes the antigenic determinant of the major human HLA histocompatibility system.

According to a published result (Brodsky, F. M., Parham, P., Barnstable, G. J., Grumpton, M. J., Bodmer, W. F., Monoclonal Antibodies for Analysis of the HLA System. Inimunol. Rev., 47:

1979; Radka, S.F., Kostyu, L.D., Amos, D.B .: A monoclonal antibody directed against the HLA-Bw6 epitope. J. Immunol., 128: 2804, 1982), it can be concluded that monoclonal antibodies specifically detecting polymorphic determinants of the HLA complex useful for diagnostic purposes can be prepared.

Substantial progress in the selection of a suitable donor in clinical organ transplantation, in the assessment of paternal spores, in the diagnosis of certain diseases that have been shown to be associated with certain HLA antigens is achieved when a monoclonal antibody producing hybridoma is available. against polymorphic determinant of HLA complex. The Bw4 hybridoma is deposited in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská 1083, under the designation HLA-01.

The hybridoma was obtained by a method known in the literature (Richiardi, P., Amoroso, A., Grepaldi, T., Ceppellini,

R., Trucco, M .: A xenogeneic monoclonal antibody recognizing. specificities controlled by HLA-A and B alleles. Immunogenetics, 12: 615-626,1981; Russo, C., Ah-Kau Ng, Pellegrino, M.A., Ferrone,

S.: The monoclonal antibody CR11-351 discriminates HLA-A2 variant identified by T cells. Immunogenetics, 18: 23-35, 1983) by cloning a set of hybrid cells obtained from the spleens of BALE / c mice immunized with lymphocytes isolated from peripheral blood of a PS donor

HLA genotype (HLA-A3.28, HLA-B14, 16/39, Bw4, Bw6, Dw2, DR2, DR6).

The advantage of hybridomas is that it produces a homogeneous monoclonal antibody that is capable of specifically reacting with cells bearing the HLA-Bw4 antigen encoded by the HLA-B locus. The HLA-01 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of BALE / c mouse mice. From cans stored in liquid nitrogen, antibody production can be started without additional antigens. Antibody pro244 399

The HLA-01 hybridoma-3 reacts specifically with human lymphocyte populations expressing the Bw4 antigen and does not need to be rid of ballast antibodies.

Example

To expand the hybridoma cells in vivo, 4 x 10 6 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 13 days of hybridoma growth in the peritoneal cavity, the mouse was exterminated and unproduced ascites fluid was collected. A total of 2.5 ml of ascites fluid was obtained which contained 6 ml of immunoglobulin. The monoclonal antibody reacted specifically in a microcytotoxic assay in the presence of complement and in a direct radioimmunoassay binding assay with peripheral blood lymphocytes expressing Bw4 antigen on its surface. In the microcytotoxic assay, the ascites fluid was still active with cells immunizing PS at a 1:10 dilution. The results of both microcytotoxic and direct radioimmunoassay showed that the monoclonal antibody HLA-01 detects the Bw4 antigen encoded by the HLA-B locus. Multiple families (8 parents and 32 children). When testing lymphocytes of 60 unrelated individuals randomly selected, the antibody reacted with lymphocytes only in humans whose lymphocytes expressed the Bw4 antigen. In family studies conducted in 8 parents and 32 children, the antigen gene detected by this antibody has been shown to segregate with the main human HLA histocompatibility system and detect the Bw4 antigen. In contrast, negative results with the monoclonal antibody HLA-01 in both assays used were obtained with Daudi cells, a permanent lyrafoid line that does not express HLA-A, -B, -C antigens as well as lymphocytes of 14 inbred mouse strains.

HLA-01 hybridoma cells have an ultrastructural pattern of typical myeloma cells. Cytoplasma contains a large number of polyribosomes, mitochondria and poorly developed endoplasmic reticulum. In vitro

244 399 grow 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). This medium (known as H-MEMd, 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 (Bioveta, Ivanovice) on Haná, $ 10). The hybridoma is cultured at 37 ° C. The mean generation time is 15.6 h and 5 months after construction, the modal number of chromosomes was 96. The antibody produced is monoclonal immunoglobulin of IgG2b subclass with light chains of kappa type, its isoelectric point is pH 6.9 «7.0.

The monoclonal antibody produced by the hybridoma HLA-01 reacts specifically with human lymphocytes isolated from peripheral blood. The HLA-01 hybridoma can be used industrially as a source of a monoclonal antibody that detects Bw4 antigen-bearing cells in diagnostic methods. The HLA-01 hybridoma monoclonal antibody can be used in clinical immunology laboratories to select a suitable donor in clinical transplants of vital organs and tissues, in the diagnosis of diseases that have been shown to be associated with HLA-Bw4 antigen and in forensic medicine in paternal disputes.

Claims (1)

SUBJECT OF THE INVENTION Mouse IMG-CZAS1 ŘLA-Ol, a lymphocyte hybridoma producing a monoclonal antibody of subclass IgQ2b against the membrane antigen of human HLA-Bw4 positive cells.