CS235850B1 - Mousy lymphocytare hybridome producing anti-matter against human transferine - Google Patents

Abstract

The invention relates to mouse lymphocytic antibody-producing hybridone against human transferrin, deposited in collection of hybridomas of the Institute of Molecular Ge - netics of the Czechoslovak Academy of Sciences under the name HTP - 04. Hybridone monoclonal antibody alone HTP-04 is suitable for use in enzyme immunoassays and radioimmunoassay and visualizing receptors for transferrin at the optical and electron levels microscope. A mixture of HTP 4, HTP 06 and HTP 14 antibodies it is applicable to precipation tests.

Description

The invention relates to a novel hybridoma, ie, a hybrid unicellular organism, constructed by fusing a mouse myeloma cell line P3-X63-Ag.0653 and a mouse spleen lymphoid cell producing an anti-human transferrin antibody.

To date, anti-transferrin antibodies have been produced by repeatedly injecting transferrin as an antigen into test animals, most commonly rabbits and pigs. The serum of the immunized animals collected after a certain period of antigen treatment serves as a source of antibodies, used in particular for the quantitative determination of plasma transferrin by simple radial immunodiffusion. This procedure, called conventional immunization, has several disadvantages. There is a heterogeneous mixture of antibodies in the serum of the immunized animals. whose spectrum is different and unique 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 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. 1003, is available IMG CZAS HTF-04.

Said hybridoma was obtained according to the method known in the literature (Fazekas de St.Groth, S, Scheidigger, D: Production of monoclonal antibodies: Stratedy and tactics, 3, Immunol, Meth., 35: 1-21, 1900; Galfré, G, Howe, S, C, Milstein, C, Butcher, G.W, Howard, 3.C,: Antibodies to major histocompatibility antigens produced by hybrid cell line, <· 3 -

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Nature 266: 550, 1977 / cloning a set of hybrid cells resulting from the fusion of murine myeloma cell 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. HTF-04 hybrids can be cultured in vitro in animal cell media and adapted for growth in vivo in the peritoneal cavity of BALB / c mouse mice. From preserves stored in liquid nitrogen, antibody production can be initiated without additional antigen. The antibody produced by the HTF-04 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 5 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) 16 days prior to transfer of the hybridoma cells. After 14 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the produced assistant fluid collected. A total of 4.Θ ml of ascites fluid containing 12 mg / ml immunoglobulins was obtained. The compound reacted with a specific antigen in an enzyme-immunoassay (using a 7-horseradish peroxidase labeled porcine anti-mouse antibody) up to 1:10, using indirect immunofluorescence techniques. the transferrin receptor was thus detected by the antibody thus obtained on human myeloma cells. A mixture of HTFO4, HTF 06 and HTF 14 antibodies is useful for precipitation assays.

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HTF-04 hybridoma cells have an ultrastructural pattern of typical myeloma cells, where the predominant organelle is free and membrane-bound polybrososomes, growing 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 (3 mM), sodium pyruvate (1 mM). This medium (referred to as H-MEMd, CSAV's Molecular Genetic Status) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (0.05 mM) HEPES buffer (10 mM) and inactivated bovine serum (Bioveta) for HTF-04 hybridoma culture. Ivanovice na Hané, 10%. The hybridoma is cultured at 37 ° C. The mean generation time is 13.1 h and 12 months after construction, the modal number of ohromosomes was 82. The antibody produced is a monoclonal immunoglobulin of subclass IgG1, kappa type,

The monoclonal antibody produced by the hybridoma HTF-0 reacts specifically with human transferrin,

The HTF-04 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-04, producing a monoclonal antibody of IgG1 subclass against human transferrin.