CS238780B1 - Mouse-type lymphocytic hybridom img-czas htf %1 - Google Patents

Abstract

The solution relates to mouse lymphocyte antibody-producing hybridoma human transferrin deposited in the collection hybridomi. Institute of Molecular Genetics CSAV under the name HTF-01. Monoclonal the hybridoma antibody HTF-01 is suitable for use in enzymatic and radioimmunological analysis.

Description

The invention relates to a novel hybrid, 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 into test animals, most commonly 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 generates, in addition to antibodies to the desired antigen or antibodies to the impurities of the antigen preparation, these must be removed from the sera by saturation. Production batches of conventional sera are therefore 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 hybridone producing a monoclonal antibody against human transferrin, deposited in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská Street, 1,083, is available IMG CZAS HTF-01.

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, S.C., Milstein, C., Butcher, G.W., Howard, J.C .: Antibodies to Major Histocompatibility Antigens Produced by Hybrid Cell Lines. 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 BAIB / c mice immunized with human transferrin.

The advantage of hybridomas is that it produces a homogeneous antibody, a monoclonal antibody that is capable of specifically reacting with human transferrin. The HTF-01 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-01 hybridoma reacts only with transferrin from human serum proteins and does not need to be rid of ballast antibodies. In addition to human transferrin, HTF-01 also reacts with equine and canine transferrin.

Example

In order to propagate hybridoma cells in vivo, 2 x 10? cells into the peritoneal cavity of mice. To improve 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 21 days of hybridoma growth in the peritoneal cavity, the mouse was killed and unproduced ascites fluid was collected. A total of 3.6 ml of ascites fluid containing 8 mg / ml immunoglobulin was obtained. The antibody reacted with a specific antigen in an enzyme-immunoassay (using a horseradish peroxidase labeled porcine anti-mouse antibody) up to a 1:10 dilution.

HTF-01 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 for cultivation of HTF-01 hybridoma, Ivanovice na Hané, 10%. The hybridoma is cultured at 37 ° C. The mean generation time is 14.9 h and 12 months after construction, the modal number of chromosomes was 84. The antibody produced is a monoclonal immunoglobulin of subclass IgG1, kappa type.

The monoclonal antibody produced by the hybridoma HTF-01 reacts specifically with human transferrin.

The HTF-01 hybridoma can be industrially used as a source of monoclonal anti-transferrin antibody in analytical or preparative methods.

Claims (1)

SUBJECT OF THE INVENTION Mouse lymphocyte hybridoma IMG CZAS HTF-01, producing a monoclonal antibody of subclass IgG1 against human transferrin.