CS239800B1 - Mouse lymphocytic hybridoma IMG CZAS TU-04 - Google Patents

Abstract

The solution concerns the mouse lymphocyte hybridoma IMG CZAS TU-04, producing an antibody against tubulin, especially porcine, deposited in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation TU-04. The monoclonal antibody of the TU-04 hybridoma itself is suitable for use in enzyme-immunological and radioimmunological analysis and in the visualization of microtubules in cells at the optical and electron microscope level.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by cell fusion of a mouse myeloma line P3-X63-Ag8.653 and a mouse spleen lymphoid cell producing an anti-swine tubulin antibody.

To date, anti-tubulin antibodies have been produced by repeatedly injecting tubulin as an antigen into test animals, most commonly rabbits. The serum of the immunized animals, taken after a period of antigen treatment, serves as a source of antibodies used, in particular, for the identification of tubulin-containing cell structures.

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.

In addition to the antibodies to the desired antigen 1, the organism will generally produce antibodies against the impurities of the antigen preparation.

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 substantially pure immunization antigen and other antigens to saturate the ballast antibodies against the 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 porcine tubulin, deposited in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská ul. 1083, is available IMG CAS TU-04 .

Said hybridoma was obtained by a method known in the literature [Fazekas de, St.,

Groth, S., Scheidigger, D .: Production of Monoolonal Antlbodies: Strategies and Taotics, J. Immunol. Meth., 35: 1-21, 1980. Galfré, G., Howe, S.C., Milstein, C., Butoher, G.W. Howard, JC: Antibodies to major histocompatibility antigens produced by hybrid cell line, Nature 266-550, 1977 / cloning a set of hybrid cells resulting from the fusion of murine myeloma line P3-X63-Ag8.653 and cells obtained from the spleen of Balb / o mice immunized with porcine tubulin.

The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is able to react specifically with porcine tubulin but also with tubulin of many other species (including human tubulin, among others).

The TU-04 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of Balb / c mice. From cans stored in liquid nitrogen, antibody production can be started without additional antigen.

The antibody produced by the TU-04 hybridoma is tubulin-specific and does not need to be rid of ballast antibodies.

Example

To expand 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 mouse was treated with paraffin oil (0.5 ml intraperitoneally) 13 days prior to transfer of the hybridoma cells. After 18 days of hybridoma growth in the peritoneal cavity, the mouse was killed and unproduced asoitic fluid collected.

A total of 3.5 ml of ascites fluid containing 15 mg / ml of 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.

Using the indirect immunofluorescence technique, the thus obtained antibody was imaged by microtubular complex in cells of mouse fibroblastoid lineage C ^ H origin. TU-04 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 and supplemented with non-essential immunoacids, 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 TU-04 hybridoma. , Ivanovice na Hané. ’

The hybridoma is cultured at 37 ° C. The mean generation time is 14 hours and 12 months after construction, the modal number of chromosomes was 79. The antibody produced is a monoclonal immunoglobulin of the IgG kappa class directed against an epitope on the alpha-subunit of tubulin.

It reacts with an epitope other than MP TU-01, as determined by immunoblotting antibodies to alpha and beta tubulin separated by SDS PAGE and to peptide fragments of whole tubulin equally separated.

The monoclonal antibody produced by the TU-04 hybridoma, in analogy to conventional antibodies, reacts with tubulin of many species. It is therefore useful for detecting cellular cytoskeletal structures of most tubulin containing species.

The TU-04 hybridoma can be used industrially as a source of anti-tubulin monoclonal antibody in analytical or preparative methods.

Claims (1)

Mouse lymphocyte hybridoma IMG CZAS TU-04, producing a monoclonal antibody of the IgM class against tubulin of many species.