CS228858B1 - Mouse lymphocytic hybridom producing an antisubstance against hog g-immunoglobulin - Google Patents

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by cell fusion of a mouse myeloma line P3-X63-Ag8.653 and a mouse spleen lymphoid cell producing an antibody against porcine immunoglobulin G.

To date, anti-immunoglobulin G antibodies have been produced by repeatedly injecting immunoglobulin G as an antigen in test animals, most commonly in rabbits or horses. The serum of the immunized animals collected after a period of antigen treatment serves as a source of antibodies used primarily for qualitative or quantitative analysis of immunoglobulin in research and immunodiagnostic practice. This procedure, called conventional immunization, has several disadvantages. In the serum of immunized animals there is a heterogeneous mixture of antibodies whose spectrum varies and does not repeat in each individual organism. As a rule, the organism will produce antibodies to the impurities of the antigen preparation in addition to the antibodies to the antigen of interest; these must be removed from the sera by saturation. Consequently, batches of conventional sera are difficult to standardize and are based on production in a wide quality range. For the manufacture of each batch, a pure immunization antigen and other antigens to saturate the ballast antibodies against impurities should be prepared.

These drawbacks of the above-mentioned and previously used procedure will be eliminated if a hybridoma producing a monoclonal antibody against prosciutic immunoglobulin G, available in the collection of hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská No. 1083, is available IMG CZAS PGG-05.

The hybridoma was obtained by a method known in the literature (Fazekas de St. Groth, S., Scheidigger, D .: Production of monoclonal antibodies: Strategies and tactics, J. Immunol. Meth., 35: 1-21, 1980; G., Howe, SC, Milstein, C., Butcher, GW, Howard, C .: Antibodies to major histocompatibility antigens produced by hybrid cell line, Nature 266: 550, 1977) by cloning a collection of hybrid cells resulting from myeloma mouse fusions line P3-X63-Ag8.653 and cells obtained from the spleen of Balb / / c mice immunized with porcine immunoglobulin G.

The advantage of the hybridoma is that it produces a homogeneous antibody, a monoclonal antibody that is capable of specifically reacting with porcine immunoglobulin G. The PGG-05 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of mice Balb strains / c. From cans stored in liquid nitrogen, antibody production can be started without additional antigen.

Example

In order to propagate hybridoma cells in vivo, 1.5 X 10 6 cells were injected into the peritoneal cavity of mice. To enhance attachment of the cells applied, the mouse was treated with paraffin oil (0.4 ml intraperitoneally) 15 days prior to transfer of the hybridoma cells. After 18 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the produced ascites fluid collected. A total of 6.0 ml of ascites fluid containing 9 mg / ml immunoglobulin was obtained. The ascites fluid containing the PGG-05 hybridoma product was mixed in equal volumes with the ascites fluid containing the PGG-02 hybridoma product. The mixture of these two monoclonal antibodies provided a precipitate with porcine immunoglobulin G in a double immunodiffusion in an agar gel.

PGG-05 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 and supplemented with non-essential amino acids, L-glutamine (3 mMj, sodium pyruvate (1 mMj). This medium (referred to as H-MEMd, CSAVJ molecular genetics status) is for PGG hybridoma cultivation -05 supplemented with penicillin, streptomycin, gentamycin, 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. , 6 hours and 8 months after construction, the modal number of chromosomes was 87. The antibody produced is a monoclonal immunoglobulin of IgG1 subclass.

The monoclonal antibody produced by the PGG-05 hybridoma provides, in admixture with the monoclonal antibody produced by the PGG-02 hybridoma, a precipitate with porcine immunoglobulin G as well as a porcine immunoglobulin G Fc fragment. The Fc fragment obtained by limited enzymatic hydrolysis of trypsin The Fc fragment contains only the constant portions of the χ chain that characterize the G class immunoglobulin. Fc fragment precipitation is evidence that the monoclonal antibody produced by the PGG-05 hybrid is directed against the antigenic determinant located in the portion of the immunoglobulin molecule forming the Fc fragment. .

The PGG-05 hybridoma can be used industrially as a source of anti-porcine immunoglobulin G antibody in analytical or preparative methods. Because the antibody is specific for a constant portion of an immunoglobulin G heavy chain, it can serve to identify and quantitatively analyze a G-class immunoglobulin. In admixture with the monoclonal antibody PGG-02, it can be used for quantitative analysis by the radial immunodiffusion method in the gel. Another possible use is in the function of the second antibody, i.e., the piOti antibody, in immunochemical and immunological methods where the first antibody is a conventional antibody prepared by immunization of test pigs. In immunohistological and immunohistochemical methods using optical or electron microscopy, a monoclonal antibody is used alone after conjugation with fluorescent substances, enzymes, ferritin or biotin. The monoclonal antibody PGG-05 is used in admixture with the monoclonal antibody PGG-02 to generate a precipitate with a second antibody, for example in a radioimmunoassay, or to form a turbidity, for example, in immunoelphelometry.

Claims (1)

Mouse lymphocyte hybridoma IMG CZAS PGG-05, producing monoclonal antibodies of the invention to a subclass of IgG1 against porcine immunoglobulin.