CS223535B1 - Mice lymfocytarous hybridon - Google Patents

Abstract

The invention relates to mouse lymphocyte antibody-producing hybridoma against a table, especially a pig, deposited in the collection of hybridomas of the Molecular Institute genetics of CSAV under the designation TU-01. Hybridoma monoclonal antibody alone TU-01 is suitable for use in enzyme immunoassays and radioimmunoassays and visualizing microtubules in cells optical and electron level microscope.

Description

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

To date, anti-tubulin antibodies have been made to be _; and ulin repeatedly injected as antigen to test animals, most commonly to rabbits. The serum of animals thus immunized, 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 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 antibodies to the desired antigen; these must be removed from the sera in a complicated way, called saturation. Consequently, production batches of conventional sera are difficult to standardize and are based on production over a wide quality range.

For the production of each batch, a substantially pure immunization antigen and other antigens to saturate the ballast antibodies against 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 is available, deposited in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská ul. 01.

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Said hybridoma was obtained by a method known in the literature (Pazekas de, -St., Groth, S., Scheidigger, D .: Production of Monoclonal Antibodies: Strategies and Tactics, J. Imiaunol. et al., 35: 1-21, 1980; Galfré, G. ILOW SG Milstein, G _e, Butcher, GU, Howard JC Antibodies that histočompatibili the major antigens produced by hybrid cell lines, Nature, 266x550,

1977 / cloning a set of hybrid cells resulting from the fusion of mouse myeloma line P3-X63-Ag8.603 and cells obtained from the spleen of Balb / c 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). hybridoma TU-01 is possible

To cultivate in vitro in animal cell media and is adapted for in vivo growth in the peritoneal cavity of Balb / c mice. Preserves stored ⁱⁿ liquid nitrogen can be used to start antibody production without additional antigen. The antibody produced by the TU-01 hybridoma is tubulin-specific and does not need to be cleared of ballast antibodies.

Example:

To expand hybridoma cells in vivo, 1 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) 9 days prior to transfer of the hybridoma cells. After 14 days of hybridoma growth in the peritoneal cavity, the cape was killed and the produced ascites fluid collected. A total of 5.0 ml of ascites fluid containing 9 µg / ml of immunoglobulin was obtained. The antibody was 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 imonofluorescence technique, the antibody thus obtained imaged a microtubular ‘complex in cells of the mouse fibroblastoid lineage G < H > H.

TU-01 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free

223 S35 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 amino acids, L-glutamine (3 mM), sodium pyruvate A mM). 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 AO mM buffer and inactivated bovine serum for cultivation of TU-01 'hybridoma. Ivanovice na Rané, 10%. The hybridoma is cultured at 3 µg. The mean generation time is 13 * 1 h and five months after construction, the modal number of chromosomes was 82. The antibody produced is a monoclonal immunoglobulin of IgG1 subclass.

The monoclonal antibody produced by the TU-01 hybridoma, in analogy to conventional antibodies, reacts with tubulin of the second-line. hů. It is therefore useful for detecting cellular cytoskeletal structures of most tubulin containing species.

The TU-01 hybridoma can be used industrially as a source of monoclonal antibody against tubulin in analytical or pre-parental methods.

Claims (1)

IMG GAS TU-01 murine lymphocyte hybridoma producing a monoclonal antibody of subclass IgG1 against tubulin, particularly porcine.