CS243348B1 - Mousy lymphocytoid hybridom img-czas hft-21 - Google Patents

Abstract

The present invention relates to mouse lymphocytic antibody-producing hybridoma human and rabbit transferrin deposited in the collection of hybrids of the Molecular Institute genetics of the CSAV under the name HTF-21. Alone HTF-21 hybridoma monoclonal antibody it is suitable for use in enzyme immunoassays and radioimmunoassay.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusing a mouse cell of the myeloma line P3-X63-Ag. 8653 and murine spleen lymphoid cells 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 advantages. 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.

The above-mentioned drawbacks of the above-mentioned and hitherto 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, Vídeňská ul. .

Said 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, Milstein, C., Butcher, GW, Howard, JC: Antibodies to major histocompatibility antigens produced by hybrid cell line, Nature 266: 550, 1977 / cloning a set of hybrid cells resulting from mouse cell fusion P3-X63-Ag myeloma lines 8.653 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-21 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 HTF-21 hybridoma reacts specifically with human and rabbit 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-mice. To improve the attachment of the cells applied, the mouse was treated with paraffin oil (0.5 ml intraperitoneally) 10 days prior to transfer of the hybridoma cells.

After 16 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the produced ascites fluid was collected. A total of 4.8 ml of ascites fluid was obtained which contained 16 mg / ml immunoglobulin.

The antibody was reacted with a specific antigen in an enzyme-immunoassay (using a horseradish peroxidase labeled porcine anti-mouse antibody) up to a 1: 10 ® dilution. The HTF-21 antibody cross-reacts with rabbit transferrin up to a 1: 10 dilution.

It does not react with the desired human serum protein in the binding assays. The HTF-21 antibody does not react with transferrin bound to the cell transfer receptor in the indirect immunofluoresoence assay using a commercial porcine anti-mouse immunoglobulin antibody (SWAM-FITC, SEVAC).

HTF-21 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 supplemented with non-esenoic amino acids, L-glutamine (3 mM), sodium pyruvate (1 mM). This medium (known as H-MEMd, Institute of Molecular Genetics of the Czechoslovak Academy of Sciences) is supplemented with penicillin, streptomyoin, gentamyoin, 2-mercaptoethanol (0.05 mM), HEPES buffer (10 mM) and inactivated bovine serum for cultivation of HTF-21 hybridoma (Bioveta, Ivanovice) in Haná, 10%.

The hybridoma is cultured at 37 ° C. The mean generation time is 12.8 hours and 12 months after Construction, the modal number of chromosomes was 81. The antibody produced is a monoclonal immunoglobulin of the IgG1 subclass with light chains of the kappa type, its isoelectric point being pi 6.8-7.1.

The monoclonal antibody produced by the HTF-21 hybridoma reacts specifically with human and rabbit transferrin.

The HTF-21 hybridoma can be industrially used as a source of anti-transferrin monoclonal antibody useful for detection of transferrin in analytical (enzymoimmunological), or preparative (uptake of transferrin from serum and other body fluids and affinity chromatography washes on immobilized antibody).

Claims (1)

SUBJECT OF THE INVENTION Mouse lymphocyte hybridoma IMG CZAS HTF-21, producing a monoclonal antibody of IgG1 subclass against human and rabbit transferrin.