CS243652B1 - Mouse lyaphocyte hybrid · IMG CZAS HP-03 - Google Patents

Abstract

Mouse lymphocyte hybridoma producing an antibody against horseradish peroxidase, deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation HP-03. The monoclonal antibody of the hybridoma HP-03 is suitable for use in enzymatic and radioimmunological analyses and in the preparation of peroxidase-antiperoxidase complexes.

Description

(54) Mouse lymphocyte hybrido · IMG CZAS HP-03

Mouse lymphocytic hybrido, producing an anti-horseradish peroxidase antibody, deposited in the Hybridoa Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the name · HP-03. The monoclonal antibody of the HP-03 hybridoma is suitable for use in enzymatic and radioimmunoassay and M preparations of the peroxidase-antiperoxidase complex.

The present invention relates to a novel hybridoma, i.e., a hybrid unicellular organism, constructed by fusing a mouse myeloma line P3-X63-Ag6.653 cell line and a murine sclerosis lymphoid cell producing an anti-horseradish peroxidase antibody.

To date, anti-horseradish peroxidase antibodies have been produced by repeatedly injecting horseradish peroxidase as an antigen in test animals, most commonly in rabbits and pigs. Serum of such immunized animals, taken after a certain period of antigen treatment, serves as a source of antibodies used mainly for the preparation of the peroxidase anti-peroxidase complex used to detect antigens, particularly in tissue sections.

This procedure, called conventional immunization, has several drawbacks. In the serum of immunized animals ae finds a heterogeneous mixture of antibodies whose spectrum is different and unrepeatable in each individual organism.

As a rule, the body produces antibodies to the desired antigen in addition to antibodies to the desired antigen: these must be removed from 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 lot, it is necessary to prepare a pure immunization antigen and other antigens to saturate the ballast antibodies against impurities.

The above drawbacks of the above-mentioned and so far used method will be eliminated if a hybridoma producing monoclonal antibody IgO1 against horseradish peroxidase, deposited in the hybridoma collection of the Institute of Molecular Genetics fiSAV in Prase 4, Vídeňská 1083, is designated IMG CZAS HP-03.

The hybridoma was lysed in a manner known from the literature (Pazekas de St. Qroth, S., Scheidigger, D .: Production of monoclonal antibody strategies and tactics, J. Immunol. Meth., 35: 1-2), 1980; Glafré, G., Howe, S.C., Milstein, C., Butcher, G.W., Howard, J.C .: Antibodies to Major Histocompatibility Antigens Produced by Bybrid Cell Lines.

Nature 266: 550, 1977) cloning a set of hybrid cells resulting from the fusion of cells of the mouse myeloma line P3-X63-Ag8.653 and cells obtained from the spleen of mouse strain C57B1 / 10 (B10) immunized with horseradish peroxidase.

The advantage of hybridomas is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is able to react specifically with horseradish peroxidase. the HP-03 hybrid can be cultured in vitro in animal cell media and is adapted for growth in vivo in the paritoneal cavity of mouse F (BlOxBALB / c).

preservatives, stored in liquid nitrogen, can be used to start antibody production without additional angtigan. The antibody produced by the HP-03 hybridoma reacts specifically with horseradish peroxidase to form a stable complex useful for antigen detection in ansymoimmunological assays.

Example

In order to propagate hybridoma cells in vivo, 2 × 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) 14 days prior to transfer of the hybridoma cells.

After 16 days of hybridoma growth in the peritoneal cavity, the unproduced asdetic fluid was also killed. In total, 4.6 ml of an acitiative fluid containing 13 mg / ml of immunoglobulin was recovered.

Monoclonal antibody reacted specifically aa sntigenam in ansymoiaunologiekém assay (using antimyáí pig antibody labeled with horseradish peroxidézou) up to a dilution of 1: 10 ^7th

HP-03 hybridoma cells find an ultrastructural image 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-glutaain (3 ait), sodium pyruvate (1 mile). This medium (referred to as H-MElid) is supplemented with HP-03 hybridoma penicillin, streptomycinine, gentamycinine, 2-aercapthoethanol (0.05 µL) HEPES buffer (10 µU) and inactivated bovine gloom (10%).

the hybrid is cultured at 37 ° C. The mean generation time is 11.4 h and 3 months after construction, the modal number of chroaosoa was 83. The antibody produced is monoclonal IgA1 subclass, pI 6.6 to 7.2.

The monoclonal antibody produced by the HP-03 hybridoma reacts specifically with horseradish peroxidesis.

Hybridoa (HP-03) can be used industrially as a source of monoclonal antibody against horseradish peroxidase in analytical, preparative methods or for the preparation of peroxidase-anti peroxidase coaplex.

Claims (1)

SUBJECT OF THE INVENTION Mouse lymphocyte hybridoa IMG CZAS HP-03 producing an aonoclonal antibody of IgG1 subclass against horseradish peroxidase.