CS245849B1 - Mouse lymphocyte hybridom img czas 6-01 - Google Patents

Abstract

The solution relates to mouse lymphocyte hybridoma, anti-vimentin antibody and desmin of a number of species deposited in the Collection of Hybridomas of the Institute of Molecular Genetics CSAV under the designation VI-01. Alone monoclonal antibody hybridoma VI-01 is Suitable for use in Enzyme Immunology radioimmunoassay of test tissues and cells, and visualizing medium filaments in cells at the optical and electron levels microscope.

Description

(54) Mouse Lymphocyte Hydridoma IMG CZAS VI-01

2

The present invention relates to a murine lymphocyte hybridoma producing an anti-vimentin antibody and desmin of a number of species deposited in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation VI-01. The monoclonal antibody of hybridomas VI-01 itself is suitable for use in enzymatic and radioimmunological analysis of test tissues and cells and visualization of medium filaments in cells at the optical and electron microscope levels.

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 antibody against porcine vimentin and desmin.

To date, anti-vimentin and desmin antibodies have been made by repeatedly injecting vimentin or desmin as an antigen in test animals, most commonly in rabbits. The serum of the animals thus immunized, taken after a period of antigen treatment, serves as a source of antibodies used, in particular, to identify cellular structures containing vimentin and desmin. 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 will produce antibodies to the impurities of the antigen preparation in addition to the antibodies to the desired autigen; these must be removed from sera in a complicated way, called 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 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 vimentin and desmin deposited in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská ul. 01.

The hybridoma was obtained by a method known in the literature (Fazekas de, St., Groth, S., S. Scheidegger, O .: Product of monoclonal antibodies: Strategies and tactics, J. Immunol. Meth., 35: 1-21, 1980, Galfré, G., Howe, SC, Milstein, C., Butcher, GW, Howard, JC: Antibodies to major histocompatibility antigens produced by hybrid cell line, Nature 266-550 (1977) myeloma lines P3-X63-Ag8. .653 and cells obtained from the spleen of B10.A mice immunized with partially purified pig brain extract.

The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is able to specifically react with vimentin and desmin of many species. VI-01 hybridoma can be cultured in vitro in animal cell media and is adapted for in vivo growth in the peritoneal cavity of first filial generation mice between BALB / c and B10.A strains. From cans stored in liquid nitrogen, antibody production can be started without additional antigen. The antibody produced by the VI-01 hybridoma is specific for vimentin and desmin only and does not react with any other intermediate filament protein and other cytoskeletal proteins as determined by immunoblotting, immuno-radiometric assay and indirect immunofluorescence. Detection was obtained by electrophoretic transfer of various cell lysates and isolated cytoskeleton preparations from the brain obtained by separation on polyacrylamide gel electrophoresis in dodecyl sulfate environment under reducing conditions. After transfer to the nitrocellulose membrane, vimentin and desmin were specifically detected using a VI-01 antibody, a porcine anti-mouse immunoglobulin antibody and a peroxidase-antiperoxidase complex (PAP) using a horseradish peroxidase monoclonal antibody. The result was further confirmed in an immunoradiometric assay, which demonstrated binding of VI-01 to desmin and vimentin and inactivity with tubulin and bovine serum albumin. Indirect immunofluorescence in a light microscope using a fluorescein-iso-thiocyanate-labeled porcine antibody against mouse immunoglobulins (Ostav sera and vaccines, Prague), only the intermediate filaments bound to the cell types tested. After colcemid treatment, a typical cluster of filaments to the nucleus occurred.

Example

To expand the 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 mouse was treated with paraffin oil (0.5 ml intraperitoneally) 13 days prior to transfer of the hybridoma cells. After 20 days of hybridoma growth in the peritoneal cavity, the mouse was killed and unproduced ascites fluid was collected. Total yield 3.0 ml scitické liquid containing 10 mg / ml (vimentin) in enzyme immunoassay (using porcine anti-mouse antibody labeled with horseradish peroxidase), up to a dilution of 1: 10 ^B. The specificity of the obtained antibody was tested on a panel of cells from tissue cultures, both by indirect immunofluorescence (using a porcine anti-mouse immunoglobulin labeled with fluorescein-iso-thiocyanate, serum and vaccine constitution, Prague) and after electrophoretic transfer of cell lysates to nitrocellulose membrane and specific detection by indirect immunoassay (see above) .The results are summarized in Table 1.

Table 1

Reactivity of VI-01 antibodies with cells of different lines (+ strong positivity, - negative result, IF - immunofluorescence, IB - immunoblotting, V - vimentin, K - keratin, G - glial acid protein, D - desmin).

Species Tissue origin, Line name Type of center- Reactivity cell type nich fila- ment IF IB Human pulmonary fibroblasts LEP V + + | adenocarcinoma HELA V + K + + bladder cancer T-24 K - - T-lymphoblastoid line MOLT-3 v + + Rat glioma C 6 V + G + + Mouse fibroblast 3T3 V + + primary culture Leydigovy V + + cells hamster ovarian cells CHO v + + kidney cells BHK-21 V + D + + From the results it is clear that the antibody and membrane-bound polyribosomes.

It brazes the vimentin type of medium filaments (Hela cells also contain keratin type, which is not shown), a number of species. Specificity was further refined in immunoblotting after transfer of purified materials to nitrocellulose (Table 2).

Table 2

Specificity of VI-01 antibody in immunoblotting after transfer of purified and partially purified materials to nitrocellulose membrane. (+ positive reaction, - negative reaction) (+ positive reaction, - negative reaction)

Material Result

Vimentin +

Desmin +

Cytokeratins -

Cytoskeletal proteins isolated from mouse brain -

Again, the reaction was only confirmed with vimentin and desmin and non-reactivity with cytokeratins (isolated from human skin), neurofilament proteins, and glial fibrillary acid protein.

VI-01 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free growing 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, the 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 germ (Bioveta) , Ivanovice na Hané, 10%). The hybridoma is cultured at 37 ° C; The mean generation time is 15.6 h and 6 months, after construction, the modal number of chromosomes was 84. The antibody produced is an IgM kappa class monoclonal immunoglobulin directed against epitol found on vimentin and desmin.

The monoclonal antibody produced by hybridoma VI-01 analogously to conventional antibodies reacts with vimentin and desmin of a number of species. It is therefore useful for the detection of cellular cytoskeletal structures of most species containing vimentin and desmin. It can be used especially in everyday work in all departments of pathological anatomy and oncology as an important aid in diagnosis. It is useful for differential diagnosis between tumors of epithelial (carcinoma) and mesenchymal (sarcoma) origin.

Claims (1)

SUBJECT OF THE INVENTION Mouse lymphocyte hybridoma IMG CZAS VI-01, producing a monoclonal antibody of the IgM class against vymentine and desmin of a number of species.