CS228860B1 - Mouse lymphocytic hybridom producing an antisubstance a - Google Patents

Description

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 human transferrin.

To date, anti-transferrin antibodies have been produced by repeatedly injecting transferrin as an antigen in test animals, most commonly in rabbits and pigs. The serum of the immunized animals collected after a period of antigen treatment serves as a source of antibodies, used in particular for the quantitative determination of plasma transferrin by a simple radial immunodiffusion method. 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 are required to saturate the ballast antibodies against impurities.

These drawbacks of the above-mentioned and previously used procedure will be eliminated if a hybridoma producing a monoclonal antibody against human transferrin, deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vienna No. 1083, is available IMG CZAS HTF-06.

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); Galfré, G., Howe, SC, Butcher, GW, Howard, JC: Antibodies to major histocompatibility antigens produced by hybrid cell line, Nature 266: 550, 1977 J by cloning a set of hybrid cells resulting from fusion of myeloma mouse cells line P3-X63-Ag8.653 and cells derived from the spleen of Balb / c mice immunized with human transferrin.

The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with human transferrin. The HTF-06 hybridoma can be cultured in vitro in media suitable for animal cells and is adapted

228880 for in vivo growth in the peritoneal cavity of Balb / c mice. From cans stored in liquid nitrogen, antibody production can be started without additional antigen. The antibody produced by the HTF-06 hybridoma is specific to human transferrin and does not need to be rid of ballast antibodies.

Example

To expand the hybridoma cells in vivo, 2 X 10 ⁶ cells were injected into the peritoneal cavity of mice. For better attachment of the cells, mice were treated with paraffin oil (0.5 ml intraperitoneally) 14 days prior to transfer of the hybridoma cells, and after 20 days of hybridoma growth in the peritoneal cavity, the mice were killed and the produced ascites fluid collected. ml ascitic fluid, which contained 15 mg / ml immunoglobulin. the antibody reacted with the specific antigen in the enzyme immunoassay (using porcine anti-mouse antibody labeled with horseradish peroxidázouj up to a dilution of 1: 10 ^7th using indirect immunofluorescence technique was thus obtained antibody detected transferrin receptors on human myeloma cells.

HTF-06 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free and membrane-bound polyribosomes. In vitro they 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 (3 mM), sodium pyruvate (1 mM). This medium (referred to as H-MEMd, Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (0.05 mM) in HEPES buffer (10 mM) and inactivated bovine serum (Bioveta) (Ivanovice na Hané, 10%) The hybridoma is cultured at 37 DEG C. The mean generation time is 19.7 hours and 12 months after construction, the modal number of chromosomes was 85. The antibody produced is a monoclonal immunoglobulin of IgG1 subclass.

The monoclonal antibody produced by the hybridoma HTF-06 reacts specifically with human transferrin.

The HTF-06 hybridoma can be used industrially as a source of anti-transferrin monoclonal antibody in analytical or preparative methods.

Claims (1)

Mouse lymphocyte hybridoma IMG CZAS HTF-06, producing the monoclonal antibody VYNALEZA to a subclass of IgG1 against human transferrin.