CS252450B1 - Mouse lymphocyte hybrid IMG SZAS HL-40 - Google Patents

Abstract

The present invention relates to a mouse lymphocyte hybridoma producing an antibody against membrane HLA class II antigens /HLA-DR, HLA-DP/ of human cells, stored in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation IMG CZAS HL-40. The monoclonal antibody of the hybridoma HL-40 is suitable for use in enzyme-linked immunosorbent and immunofluorescence analysis of human cells with membrane HLA class II antigens; its advantage is a very pronounced labeling of target cells in suspensions and on tissue sections.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line P3-X63-Ag0.653 and a mouse spleen lymphoid cell producing an antibody against the monomorphic determinant of membrane HLA antigens II. class, ZHLA-OR, HLA-DP / present on human B cells, monocytes, macrophages and activated T cells. These antigens are essential for regulating various functions of the immune system. Quantitative determination of cells with membrane HLA antigens II. class is important in the diagnosis of some pathological conditions and the classification of human hematological cancer.

To date, antibodies to human cell membrane antigens have been made by repeatedly injecting cell suspensions or membrane fractions into production animals, most commonly rabbits. Serum of animals immunized in this way, taken after a period of antigen treatment, serves as a source of antibodies. Thus, for example, antibodies against human thymocytes are prepared which are used to inhibit allotransplantation reactions in organ transplantation in clinical medicine. This procedure, called conventional immunization, has the inevitable disadvantage that sera contain a heterogeneous mixture of antibodies against many different membrane antigens, some of which are present on multiple cell types; therefore, sera cannot 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 house hybrio product - a monoclonal antibody - is always directed against a single antigenic determinant. If a sufficiently large set of hybridomas is constructed, it is likely to find a hybrid synthesizing antibody that recognizes the antigenic determinant characterizing a particular type or class or subclass of lymphocytes.

According to the published result / Brodsky, F.M., Parham, P.,

Bodmer, W.F .: Monoclonal Antibodies to HLA-DR Determinants,

Tissue Antigens 16:30, 1980], it can be concluded that monoclonal antibodies recognizing those determinants found on HLA class II antigens, a class of all individuals in the population (monomorphic determinants), can be prepared. Such monoclonal antibodies are preferred for diagnostic use.

Substantial progress in the diagnosis of human leukocytes and diseases derived therefrom, manifested by a higher degree of analytical outcome, is achieved, when available with a hybrid, producing a monoclonal antibody against the monomorphic determinant of human HLA antigens II. class deposited in the Collection of the hypothalamic _T hybridomas Institute of Molecular Genetics, Czechoslovak Academy of Sciences 4, Vienna no. 1083, under the designation HL-IMG Czasy 40

The hybridoma was obtained by a method known in the literature. Groth, S. Scheidegger, D .: Production of Monoclonal Antibodies: Strategies and Tactics, 3. Immunol. Meth., 35: 1-21 (1980); Galfré, G., Howe, S. C., Milstein, C., Butcher, G. W., Howard, 3.C .: Antibodies to major histocompati-; bility antigens produced by hybrid cell line, Nature, 266: 550, 1977 / by cloning a collection of hybrid cells obtained from the spleen of BALB / c mice immunized with cells of the human B lymphoblastoid line of Paradise.

The advantage of the hybridoma is that it produces a homogeneous monoclonal antibody that is capable of specifically reacting with cells bearing HLA antigens II. class. The HL-40 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 cans stored in liquid nitrogen, antibody production can be started without additional antigens. The antibody produced by the HL-40 hybridoma reacts specifically with human cell populations in which HLA antigens are present in the membrane

II. class (HLA-DR, HLA-DP) and there is no need to get rid of antibodies »ballast.

- 3 Example

To expand the hybridoma cells in vivo, 3 x 10 6 cells were injected 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 11 days of hybridoma growth in the peritoneal cavity, the mouse was sacrificed and the non-produced ascites fluid was collected. A total of 5.5 ml of ascites fluid containing 16 mg / ml immunoglobulin was obtained. The monoclonal antibody reacted specifically in an indirect immunofluorescence or enzymatic immunoassay with a portion of peripheral blood lymphocytes. Both methods revealed 25-30% positive cells in the enzyme immunoassay

5 tests up to a 1: 10 dilution of ascites plasma and in the indirect test, immunofluorescence up to a 1: 10 dilution. Using purified T and B cell populations, HL-40 antibody reactivity was found to be greater than 95% B cells and less than 5% T cells. More than 95% of monocytes were also strongly positive, granulocytes and erythrocytes were negative. The specificity of the HL-40 antibody corresponds to the standard foreign monoclonal antibody RF-HLA-DR, as demonstrated by repeated parallel assays, including the use of antibody mixtures, both for a mixture of peripheral lymphocytes and isolated cell populations (see table) and stabilized human lymphoid and non-lymphoid lines.

Tab. Reactivity of antibodies produced by HL-40 and 1 RF-HLA-DR hybridoma to peripheral blood cells

Peripheral% positive cells reactive with monoclonal * 2 · 3 human blood antibody in an enzyme-immunological or indirect immunofluorescence assay HL-40 RF-HLA-DR HL-40 + RF-HLA-DR

lymphocytes 25d £ 30 5 T lymphocytes 5rf10 B lymphocytes 95 monocytes 95 granulocytes 1 erythrocytes 1

25tf30 i 25 £ 30 5®10 5rf10 95 95 95 95 1 1 1 1

The monoclonal antibody RF-HLA-DR was obtained from prof. G. Janossy of The Royal Free Hospital, London.

Human peripheral blood cells were isolated by the described method (B) yum, A. Isolation of lymphocytes, granulocytes and macrophages. Scand. 0. Immunol. 5, Suppl. 5: 9, 1976) on a Verografin Flux gradient.

The procedure of Lansdorp, PM, Astaldi, GCB, Osterhofhof, F., Janssen, MC, Zeijlemaker, WPs Immunopero _in xi-; dase procedures to detect monoclonal antibodies against cell surface antigen. Quantitation of binding and staining of individual cells. J. Immunol. Metb. 39: 393-405 (1980).

An indirect immunofluorescence technique using a fluorescein isothiocyanate labeled porcine anti-mouse immunoglobulin antibody (OSOL, Prague) was used.

The lymphocytes were isolated by passing the lymphocyte mixture through a nylon cotton column as described by Julius, M. H., Simpson, E. Herzenberg, L. A., A rapid method for isolation of functional thymus-derived murine lymphocytes, Europ. 3. Immunol., 3: 645-649, 1973].

B lymphocytes were isolated by adsorption onto 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- ímmunoglobulin. 3. Immunol. Meth. 15: 47-56, 1977].

The identity of the antigens recognized by HL-4Q and RF-HLA-DR was demonstrated by isolating the respective antigens by immunoaffinity chromatography on immobilized antibody HL-40 and demonstrating the strong reactivity of the antigens with RF-HLA-DR. The antigen obtained by Sepharose 4B immunoaffinity chromatography with the covalently bound monoclonal antibody HL-40 provided polyacrylamide gel electrophoresis in the presence of dodecyl. sodium sulphate under non-reducing conditions, a strong zone corresponding to m.h. 65,000 and weak zones of MW. 29,000 and 33,000 (sample was not heated), or only 2 zones corresponding to MW.

000 and 33 000 / sample in advance 2 min. heated to 100 [deg.] C., a typical result of HLA antigens II. class / Shackleford, D.A., Kaufnan, 3rd, 6, -. Korman, A.J., Strominger, J.L .: HLA-DR Antigensj Structure, Separation of Subpopulation, Gene Cloning and Function Immunol. Roar. B6: 133-187 (1982)]. After electrophoretic transfer of separated proteins to nitrocellulose membrane and specific detection by indirect enzyme immunoassay (using the appropriate antibody and porcine anti-mouse immunoglobulins conjugated with peroxidase // USSL, Prague), both antibodies (HL-40 and RF-HLA-DR) reacted strongly with antigen in dimeric form / mh

000 / i with a m.h. 29,000 isolated antigen.

HL-40 hybridoma cells have an ultrastructural image of typical myeloma cells. Cytoplasma contains a large number of polyribosomes, mitochondria, a weakly developed endoplasmic reticulum,

They grow in vitro as a suspension of culture. 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 Qstav 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 HL-40 hybridoma, Ivanovice na Hané. The hybridoma is cultured at 37 ° C. The mean generation time is 17.1 h and 1B months after construction, the modal number of chromosomes was 78. The antibody produced is monoclonal immunoglobulin of IgG2a subclass with light chains of kappa type, its isoelectric point is pH 7.8 4 Í 8.4.

The monoclonal antibody produced by the HL-40 i hybridoma specifically reacts with normal human B cells, monocytes, macrophages and activated T cells. The HL-40 hybridoma can be used industrially as a source of monoclonal antibody against cells with membrane HLA antigens II. class in analytical methods. The monoclonal antibody of the HL-40 hybridoma can be used for the determination of these cells in peripheral blood and lymphatic organs in clinical diagnosis in healthcare facilities, particularly in various immunological deficiencies and autoimmune diseases, and for the classification of leukemias and malignant lymphomas. The advantage of the monoclonal antibody HL-40 is the very prominent labeling of pulse cells carrying HLA antigens II. Classes (HLA-DR, HLA-DP) in immunofluorescence and enzyme immunoassays of cell suspensions and tissue sections.

Claims (1)

pReomE.t vynAlezu Mouse lymphocyte hybridoma IMG CZAS HL-40 producing a monoclonal antibody of IgG2a subclass against membrane HLA antigens II. class of human cells.