CS236001B1 - Mouse lymphocyte hybridoma producing anti-human transferrin antibody - Google Patents

Abstract

The invention relates to a mouse lymphocyte hybridoma producing an antibody against human transferrin, deposited in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation HTF-14. The monoclonal antibody of the hybridoma HTF-14 itself is suitable for use in enzyme-immunological and radioimmunological analysis. Mixtures of antibodies HTF-04, HTF-06 and HTF-14 or HTF-05, HTF-06 and HTF-14 are usable for precipitation tests.

Description

The invention relates to a novel hybridoma, i.e., a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line P3-X63-Ag, 8653 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 to test animals, most commonly rabbits and pigs. The serum of the immunized animals collected after a period of antigen exposure serves as a source of antibodies used mainly for quantitative determination of plasma transferrin by single radial immunodiffusion. This procedure, called conventional immunization, has several drawbacks. In the serum of immunized animals there is a heterogeneous mixture of antibodies whose spectrum is different and unrepeatable 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 shedding. Consequently, batches of conventional sera are difficult to standardize and are based on production in a wide quality range. For the production of each batch it is necessary to prepare pure immunization antigen and other antigens for saturation of ballast antibodies against impurities!

The above-mentioned drawbacks of the above-mentioned and so far used procedure will be eliminated if a hybridoma producing a monoclonal antibody against human transferrin is deposited in the Collection of Hybridomas of the Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská ul, 1083 under the name IMG CZAS HTF-14

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, 3, Immunol.Meth, 35: 1-21, 1980; Galfré, G, Howe, S.C, Milstein,

C., Butcher, G, W ,, Howard, 3rd C .: Antibodies to Major Histocompatibility Antigens Produced by Hybrid Cell Lines,

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Nature 266: 550, 1977 / cloning a set of hybrid cells resulting from the fusion of murine myeloma line P3-X63-Ag8.653 cells 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-14 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 preserves stored in liquid nitrogen, antibody production can be started without additional antigen. The antibody produced by the hybridoma HTF-14 reacts specifically with human and porcine 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. To improve attachment of the cells applied, the mice were treated with paraffin oil (0.5 ml intraperitoneally) 18 days prior to transfer of the hybridoma cells. After 17 days of hybridoma growth in the peritoneal cavity, the mouse was killed and unproduced ascites fluid was collected. A total of 3 - 8 ml of ascites fluid containing 8 ml / ml of immunoglobulin was obtained. The antibody reacted with the specific antigen in an enzyme-immunoassay (using a horseradish peroxidase labeled porcine anti-mouse antibody) up to a 1:10 dilution. A mixture of antibodies HTF-04, HTF-06 and HTF-14, or HTF-05, HTF-06 and

HTF-14 are useful for precipitation assays. The HTF-14 antibody blocks the binding of transferrin to the transferrin receptor and cross-reacts with porcine transferrin, and does not react with any human serum protein in binding assays.

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HTF-14 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-essential amino acids, L-glutamine (3mM), sodium pyruvate (1mM). 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) 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 12 _{r to} 0 h and 12 months after construction, the modal number of ohromosomes was Θ5.

The monoclonal antibody produced by the hybridoma HTF-14 reacts specifically with human and porcine transferrin.

The HTF-14 hybridoma can be used industrially as a source of the anti-transferrin monoclonal antibody in analytical or preparative methods.

Claims (1)

Mouse lymphocyte hybridoma IMG CZAS HTF-14, producing a monoclonal antibody of subclass IgG1 against human transferrin.