CS245849B1 - Mouse lymphocyte hydride IMG CZAS VI-01 - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma, producing an antibody against vimentin 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 the hybridoma VI-01 itself is suitable for use in enzyme-immunological and radioimmunological analysis of tested tissues and cells and in the visualization of intermediate filaments in cells at the optical and electron microscope level.

Description

(54) Mouse lymphocytic hydridoma IMG TIME VI-01

2

The solution concerns a mouse lymphocyte hybridoma, producing an antibody against vimentin 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 the hybridoma VI-01 itself is suitable for use in enzyme-immunological and radioimmunological analysis of tested tissues and cells and in the visualization of intermediate filaments in cells at the optical and electron microscope level.

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by cell fusion of the mouse myeloma line P3-X63-Ag8.653 and a mouse splenic lymphoid cell, producing an antibody against porcine vimentin and desmin.

Up to now, antibodies against vimentin and desmin have been produced by repeatedly injecting vimentin or desmin as an antigen into experimental animals, most often rabbits. The serum of animals immunized in this way, collected after a certain period of exposure to the antigen, serves as a source of antibodies, used mainly for the identification of cellular structures containing vimentin and desmin. This procedure, called conventional immunization, has several disadvantages. The serum of immunized animals contains a heterogeneous mixture of antibodies, the spectrum of which is different and unrepeatable in each individual organism. As a rule, the organism produces, in addition to antibodies against the desired autogen, antibodies against impurities of the antigen preparation; these must be removed from the serum by a complex method called saturation. Production batches of conventional sera are therefore difficult to standardize and come out of production with a wide range of quality.

For the production of each batch, it is necessary to prepare a highly pure immunizing antigen and other antigens to saturate ballast antibodies against impurities.

The above-mentioned shortcomings of the previously used procedure will be eliminated if a hybridoma producing monoclonal antibodies against vimentin and desmin is available, stored in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská ul. 1083, under the designation IMG CZAS VI-01.

The hybridoma was obtained in a manner known from the literature (Fazekas de, St., Groth, S., S. Scheidegger, O.: 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) by cloning a set of hybrid cells resulting from the fusion of the mouse myeloma line P3-X63-Ag8. .653 and cells obtained from the spleen of mice of the B10.A strain, immunized with a partially purified porcine brain extract.

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

Example

In order to propagate the hybridoma cells in vivo, 1 x 10 ⁶ cells were injected into the peritoneal cavity of mice. In order to improve the adhesion of the injected cells, the mouse was treated with paraffin oil (0.5 ml intraperitoneally) 13 days before the transfer of the hybridoma cells. After 20 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the unproduced ascitic fluid was collected. A total of 3.0 ml of ascitic fluid was obtained, which contained 10 mg/ml (vimentin) in an enzyme immunoassay (using a pig 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 in indirect immunofluorescence (using a swine antibody against mouse immunoglobulin labeled with fluorescein-iso-thiocyanate, Ostav sér a óčkovacích klátky, Prague) and after electrophoretic transfer of cell lysates to a nitrocellulose membrane and specific detection by indirect immunological test (see above). The results are summarized in Table 1.

Table No. 1

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

Type Tissue origin, Line name Type medium- Reactivity cell type their fil- ment IF IB Man lung fibroblasts LEP In + + | adenocarcinoma HELLA V + K + + bladder cancer T-24 To — — T-lymphoblastoid line MOLT-3 in + + Rat glioma C 6 V + G + + Mouse fibroblast 3T3 In + + primary culture Leydig cells In + + cells Hamster ovarian cells CHO in + + kidney cells BHK-21 V + D + + From the results it is clear that the antibody is and membrane-bound polyribosomes.

It shows the vimentin type of intermediate filaments (Hela cells also contain the keratin type, which is not shown), a number of species. The specificity was further clarified in immunoblotting after transferring purified materials to nitrocellulose (Table 2).

Table No. 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, reaction was confirmed only with vimentin and desmin and non-reactivity with cytokeratins (isolated from human skin), neurofilament proteins and glial fibrillary acidic protein.

VI-01 hybridoma cells have an ultrastructural image of typical myeloma cells, where the predominant organelles are free-floating and grow as semi-suspension cultures. The basic culture medium is Eagle's minimal essential medium with Hanks' salt mixture, supplemented with non-essential amino acids, L-glutamine (3 mM), sodium pyruvate (1 mM). This medium (designated as H-MEMd, Molecular Genetics Department of the Czech Academy of Sciences) is supplemented with penicillin, streptomycin, gentamycin, 2-mercaptoethanol (0.05 mM), HEPES buffer (10 mM) and inactivated bovine serum (Bioveta, Ivanovice na Hané, 10%) for the cultivation of VI-01 hybridomas. The hybridoma is cultivated at 37 °C; The mean generation time is 15.6 hours and 6 months, after construction the modal number of chromosomes was 84. The antibody produced is a monoclonal immunoglobulin of the IgM kappa class, directed against the epithelium found on vimentin and desmin.

The monoclonal antibody produced by the VI-01 hybridoma reacts with vimentin and desmin of many species analogously to conventional antibodies. It is therefore useful for detecting cellular cytoskeletal structures of most species containing vimentin and desmin. It will find application especially in everyday work in all departments of pathological anatomy and oncology, as an important aid in establishing a 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.