CS243879B1 - Mouse lymphocyte hybrid IMG-CZAS HL-38 - Google Patents

Abstract

Mouse lymphocyte hybridoma IMG CZAS HL-38 producing monoclonal antibody of the Ig G2a subclass against the membrane antigen of human HLA-DR positive cells.

Description

Author of the book

NEMEC MILOS RNDr. CSc. *, ANGELISOVÁ PAVLA Leader: RNDr. CSc., * WORLD VÁCLAV RNDr. CSc., PRAGUE (54) Mouse lymphocyte hybrid IMG-CZAS HL-38

IMG CZAS HL-38 lymphocyte hybridoma mice producing a monoclonal antibody of the Ig G2a subclass against the membrane antigen of human HLA-DR positive cells.

The invention relates to murine lymphocyte hybridomas producing an antibody against the membrane antigen of human HLA-DR positive cells deposited in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation HL-38. The monoclonal antibody of HL-38 hybridomas alone is suitable for use in enzymatic and immunofluorescence analysis of human HLA-DR positive cells.

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 lymphoid cell producing an antibody against the HLA-DR membrane antigen 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 HLA-DR positive cells is important in _x diagnosis of some pathological conditions.

To date, antibodies against membrane antigens have been produced by cell suspensions or membrane fractions being repeatedly injected into production animals, most commonly rabbits. The serum of the immunized animals 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 unavoidable disadvantage that sera contain antibodies against many 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 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 type or class or subclass of lymphocytes is likely to be found therein.

According to a published result (Brodsky, FM, Parham, P., Bodmer, WF: Monoclonal antibodies to HLA-DR determinants, Tissue Antigens 16: 30, 1980), it can be concluded that monoclonal antibodies recognizing such determinants found on HLA-DR antigens of all individuals in the population (monomorphic determinants). Such monoclonal antibodies are preferred for diagnostic use.

Substantial progress in the diagnosis of human lymphocytes, manifested by higher reliability of the analytical result, will be achieved when a hybridoma producing a monoclonal antibody against the monomorphic determinant of human HLA-DR antigens is available, deposited in the Hybridoma Collection of the Institute of Molecular Genetics 1 083, under the designation IMG CZAS HL-38.

The 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, SC, Milstein, C., Butcher, GW, Howard, JC: 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 immunized with cells of the human B lymphoblastoid lineage of Paradise.

The advantage of hybridomas is that it produces a homogeneous monoclonal antibody that is capable of specifically reacting with cells bearing HLA-DR antigens. The HL-38 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 antigen. The antibody produced by the HL-38 hybridoma reacts specifically with human cell populations in which HLA-DR antigens are present in the membrane and does not need to be rid of ballast antibodies.

Example

To multiply hybridoma cells in vivo, 6x10 6 cells were injected into the peritoneal cavity of the mouse. To improve attachment of the cells applied, the mouse was treated with paraffin oil (0.5 ml intraperitoneally) 20 days prior to transfer of the hybridoma cells. After 14 days of hybridoma growth in the peritoneal cavity, the mouse was sacrificed and unproduced ascites fluid was collected. A total of 4 ml of ascites fluid containing 15 mg / ml of immunoglobulin was obtained. The monoclonal antibody reacted specifically in an indirect immunofluorescence or enzyme-immunoassay with a portion of peripheral blood lymphocytes.

Both methods revealed 25 to 30% positive cells in the enzyme immuno-dough up to a 1: 10® ascitic plasma dilution and an indirect immunofluorescence test up to a 1: 10 dilution ⁴ . Using purified T and B cell populations, HL-38 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 HL-38 is identical to 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).

Table - Reactivity of antibodies produced by hybridoma HL-38 and RF-HLA-DR '' with peripheral blood cells

- 2 Peripheral human blood cells % of positive cells reacting with the monoclonal antibody in an enzymatic immunological or indirect immunofluo- Resistance test ^ HL-38 RF-HLA-DR HL-38 + RF-HLA-DR lymphocytes 25 to 30 25 to 30 25 to 30 T lymphocytes 5 to 10 5 to 10 5 to 10 B lymphocytes 95 95 95 monocytes 95 95 . 95 granulocytes 1 1 1 erythrocytes 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 as described (Béyum, A .: Isolatin of lymphocytes, granulocytes and macrophages. Scand. J. Immunol. 5, Suppl. 5: 9, 1976) on a VerografinFicoll gradient.

The procedure of Lansdorp, Ρ. M., Astaldi, G.C.B., Oosterhof, F., Janssen,

M. C., Zeijlemaker, W. P .: Immunoperoxidase procedures to detect monoclonal antibodies against cell surface antigen. Quantitation of binding and staining of individual cells.

J. Immunol. Meth. 39: 393-405, 1980.

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

T cells were isolated by passing a mixture of lymphocytes through a nylon cotton column as described (Julius, H., Simpson, E., Herzenberg, LA: A rapid method for isolation of functional thymus-derived murine lymphocytes. Europ. J. Immunol. 3 : 645-649 (1971).

® B lymphocytes were isolated by adsorption onto plastic plates coated with an anti-human immunoglobulin antibody as described (Mage, MG, McHugh, LL, Rothstein, TL): Lymphocytes with and without immunoglobulin surface: Preparative scale separation in polystyrene tissue culture coated with targeted anti Immunoglobulin J. Immunol.

Meth. 15: 47-56 (1977).

The identity of the antigens recognized by HL-38 and RF-HLA-DR was demonstrated by isolating the respective antigen by immunoaffinity chromatography on immobilized HL-38 antibody and demonstrating the strong reactivity of this antigen to RF-HLA-DR. The antigen obtained by Sepharose 4B immunoaffinity chromatography with covalently bound monoclonal antibody HL-38 provided, after electrophoresis in a polyacrylamide gel in the presence of sodium dodecyl sulfate, under a non-reducing condition, a strong zone corresponding to 65,000 m and weak zones of 29,000 and 33,000 mh (sample was not heated) or only 2 zones corresponding to MW 29,000 and 33,000 (pre-heated at 100 ° C for 2 min), a result typical of HLA-DR antigens (Shackleford,

D.A., Kaufman, J.F., Korman, A.J., Strominger, J.L .: HLA-DR Antigens: Structure, Separation of Subpopulation, Gene Cloning and Function. Immunol. Roar. 66: 133-187 (1982).

After electrophoretic transfer of separated proteins to nitrocellulose membrane and specific detection by indirect enzyme-immunoassay using appropriate antibody and porcine peroxidase-conjugated porcine anti-mouse immunoglobulin antibody, Prague, both antibodies (HL-38 and RF-HLA-DR) reacted strongly with the antigen in dimeric form (mh 65,000) with the mh subunit of 29,000 isolated antigen.

HL-38 hybridoma cells have an ultrastructural image of typical myeloma cells. Cytoplasm contains a large number of polyribosomes, mitochondria and poorly developed andoplasmic 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, the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin, gentamycline, 2-mercaptoethanol (0.05 mM), HEPES buffer (10 mM) and inactivated bovine serum (Bioveta) , Ivanovice na Hané 10%). The hybridoma is cultured at 37 ° C. The mean generation time is 16.2 h and 9 months after construction, the modal number of chromosomes was 85. The antibody produced is monoclonal immunoglobulin of the IgG2a subclass with light chains of kappa type, its isoelectric point is pH 5.9 to 6.5.

The monoclonal antibody produced by the HL-38 hybridoma reacts specifically with normal human B cells, monocytes, macrophages and activated T cells. The HL-38 hybridoma can be industrially used as a source of monoclonal antibody against HLA-DR positive cells in analytical methods. The monoclonal antibody of the HL-38 hybridoma can be used for the determination of HLA-DR positive 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.

OBJECT OF THE INVENTION

Claims (1)

IMG CZAS HL-38 mouse lymphocyte hybridoma producing a monoclonal antibody of Ig G2a subclass against the membrane antigen of human HLA-DR positive cells. Severography, n. P., MOST