RU1819353C - Hybridoma strain atcchb 9580 - a producer of antibodies against glycoprotein - Google Patents

Abstract

The invention relates to immunology and can be used to prepare a monoclonal antibody derived from an anti-glycoprotein. The purpose of the invention the creation of the headquarters Sptwe i ATCCNV 9580 The method consists in that the mouse is subjected to double immunization with JV cells in RP.MI medium followed by immunization with differentiated myristate acetate phorbol labels U 937 and in the same medium, the cells of the immunized immunological cells select and merge with myeloma cells of REHbZac. 8853 followed by an incubation of cells that have fused in the presence of phorbolve myristate complex aphra and JX or SKW-3 cells, a supernatant that inhibits the aggregation of JX cells. subjected to double cloying using the method of limited digestion, followed by cultivation of the cloned cells and, suitably, the medium and isolation of the final product. Yo

Description

The invention relates to the production of monoclonal antibodies against proteins, in particular to a method for producing a monoclonal antibody derived from an anti-glycoprotein.

The purpose of the invention is to provide a new monoclonal anti-glycoprotein antibody useful in the fields of organ transplantation, tissue vaccination, oncology and for the treatment of allergies.

The method is carried out as follows.

The method consists in the fact that mice are subjected to double immunization with JY cells in RPMI medium followed by immunization with U937 cells differentiated with phorbol myoistate acetate in the same

medium, spleen cells of immunized mice were selected and fused with PXbbzad 8653 myeloma cells, followed by incubation of the fused cells in the presence of phorbol myristate ester and JY or SKW-3 cells, the supernatant inhibiting JY cell aggregation was subjected to double cloning using the limited cloning method subsequent cultivation of the cloned cells in a suitable medium and isolation of the final product.

PRI me R 1. Obtaining monoclonal antibodies to ICAM-1,

Immunization Balb / C mice are immunized (intraperitoneally) with 0.5 m 2 x 107 JY cells in an RPMI medium of 103 days and 24

ABOUT

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days if not. On the 4th and 3rd days prior to fusion, mice were immunized (intraperitoneally) with 107 U937 cells differentiated with phorbol-12-myristate acetate (hereinafter FMA) in 0.5 ml of RPMI medium.

Cell differentiation 937.

5 x 105 / ml U 937 cells (ATCC CRL1593) were differentiated by incubation in RPMI medium containing 10% fetal bovine serum. 1% glutamine, 50 μg / ml gentamine and 2 ng / ml FMA, in sterile polypropylene containers. On the third day of incubation, half the volume of the medium was taken and replaced with fresh complete medium containing PMA. On the fourth day, the cells were removed, washed and prepared for immunization.

Merge.

The spleen cells from immunized mice are fused with myeloma cells RZHbZad.8653 in a ratio of 4; 1. After fusion, cells are transferred to flat-bottomed microtiter plates with 96 wells at a concentration of 105 spleen cells / well.

Selection of anti-1CAM-1 positive cells.

After a week, 50 μl of the supernatant was screened as follows. Cell lines J Y and SRW-3 were washed twice with RPMI 1640 medium containing 5 mM Gepes buffer and resuspended to a concentration of 2 x 10 cells / ml. 50 μl of the supernatant monoclonal antibody is added to 50 μl of complete RPMI medium containing 200 ng / ml phorbol ester, PMA and 100 μl of cells at a concentration of 2 x 10 cells / ml of medium. A final concentration of 50 ng / ml PMA and 2 x 10 cells per well are obtained. Cells are allowed to precipitate and the degree of aggregation at various points in time is determined. Cells were selected from supernatants incubating JY cell aggregation but not SKW-3 cell aggregation, and cloned twice using a limited dilution method. As a result of these experiments, three separate hybridoma lines that produce anti-1CAM-1 monoclonal antibodies are identified and cloned. Antibodies produced by these hybridoma lines are IgGaa, IgGab and IgM, respectively. A hybrid cell line that produces IgGza antibodies is designated R6 5 D6 T9 B2. The antibody produced by the preferred hybridoma cell line is designated P6 5 D6 E9 B2 (hereinafter R6-5-D6). The hybridoma cell line R6 5 D6 E9 B2 was deposited on October 30, 1987, in

American Typeculture Collection and designated ATCCNV 9580.

Antibody production is as follows. 3-10 x 10 6 cells are inoculated intraperitoneally with Balb / C mice. A few days prior to inoculation, the mice are intraperitoneally given either an incomplete composition of Freund's supplement or pristan. After several days, the resulting ascitic fluid was collected and the antibody was isolated by precipitation with ammonium sulfate and affinity chromatography on protein A bound to the carrier.

Example 2. The effect of anti-ICAM-1 antibody on the proliferation of human peripheral mononuclear blood cells.

Human peripheral blood mononuclear cells are induced to proliferate by the presence and recognition of antigens or mitogens. Some molecules, such as mitogen, concanavalin A, or T-cell binding antibody, OCTZ, cause nonspecific proliferation of peripheral mononuclear blood cells. Human peripheral mononuclear blood cells are heterogeneous in that they are composed of subpopulations of cells that are

0 ways to recognize specific antigens. When a peripheral blood mononuclear cell, which is capable of recognizing a specific specific antigen, encounters an antigen, a proliferation of such a subpopulation of mononuclear cells occurs. Tetanus toxin and saucer hemocyanin are examples of antigens that are recognized by subpopulations of peripheral mononuclear cells, but not

0 are recognized by all peripheral mononuclear cells in sensitized individuals.

The ability of the R6-5-16 anti-1CAM-1 monoclonal body to inhibit the proliferation of human peripheral mononuclear blood cells in systems that require interclonal adhesion is investigated.

After purification, peripheral mononuclear blood cells were washed three times with RPMI 1640 medium and cultured in flat-bottomed micronitre plates with 96 wells at a concentration of 10 cell-ml in RPM11640 medium supplemented with 10%

5 fetal bovine serum, 2 mM glutamine and 50 μg / ml heptamycin.

Antigen, or T-cell mitogen, concanavalin A (0.25 µg / ml), T-cell binding antibody OKTZ (0.001 µg / ml); a hemocyanin platter (10 g / ml) or tetanus toxoid (as a 1: 100 dilution) is added to cells that are cultured as described above in the presence of R6-5-16 in the absence of antibody (final concentration 5 g / ml) . Cells were cultured for 3.5 days (experiment with concanavalin A), 2.5 days (experiment with OKTZ) or 5.5 days (experiments with saucer hemocyanin and tetanus toxin antigen).

18 hours before the end of the experiment, 2.5 µS was added to the cultures; 3H-thymidine. Cell proliferation is determined by measuring the incorporation of thymidine into the DNA by peripheral blood mononuclear cells. The incorporated thymidine is collected and counted using a liquid scintillation counter.

The anti-ICAM-1 antibody has been found to inhibit mononuclear cells

proliferative responses to the non-specific T-cell mitogen, concanavalin A, non-specific T-cell antigen, OCT-3, and specific antigens, hemocyanin

saucer and tetanus toxoid antigen. Inhibition by anti-adhesive antibody is comparable to inhibition by anti-LFA-l antibody. Therefore, it can be assumed that ICAM-1 is a functional LFA-1 ligand and that ICAM-1 antagonism will inhibit the responses of specific defense systems.

Claims (1)

Claims The ATCCCH 9580 hybridoma strain (American Typeculture Collection Rochvill MB 20852 US A) is an anti-glycoprotein antibody producer.