CS247450B1 - Mouse lymphocytic hybridoma IMG CZAS MA-01 - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody against microtubule-associated proteins (MAP 2A and MAP 2B), deposited in the Hybridoma Collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation MA-01. The monoclonal antibody of the hybridoma MA-01 itself is suitable for use in enzyme-immunological and radioimmunological analysis, in the visualization of MAP-2 proteins in cells at the optical and electron microscope level, and for biochemical and biological analysis of the target antigenic structure.

Description

The present invention relates to a novel hybridoma, i.e., a hybrid unicellular organism, constructed by cell fusion of murine myeloma line P3-X63-Ag8.653 and murine spleen lymphoid cells producing an antibody against proteins associated with MAP-2 microtubules.

To date, antibodies to microtubule-associated proteins (MAP) have been produced by isolating MPA from brain tissue and re-injecting as antigen to test animals, most commonly rabbits. The serum of the immunized animals, taken over time with antigen, serves as a source of antibodies used to identify MAP-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 varies and does not repeat in each individual organism.

As a rule, the organism will produce antibodies to the impurities of the antigenic preparation in addition to the antibodies to the antigen of interest; these must be removed from the 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.

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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 MAP-2, deposited in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská ul. 01.

Said hybridoma was obtained by a method known in the literature (Fazekas de, St., Groth, S., Schneidigger, D .: Production of monoclonal antibodies: Strategies and tactics, 3rd 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) P3-X63-Ag8.653 and cells obtained from the spleen of Balb / c mice immunized with microtubular proteins (containing tubulin and microtubule-associated proteins) prepared from pig brain.

The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with MAP-2 associated microtubule proteins. The reactivity of MA-01 monoclonal antibody with MAP-2 proteins has been demonstrated in immunoblotting (protein separation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate under reducing conditions, subsequent electrophoretic transfer of proteins to nitrocellulose membrane, specific detection by indirect immunoassay using MA-01). peroxidase / mouse monoclonal antibody against peroxidase (Viklicky V., Dráber P., Macků B., Dráberová E., Rozprimová L. 1984 Fol. biol. Prague 30, 377-382). high molecular weight proteins found at the position of the reactive molecular weights of the corresponding MAP-2A and MAP-2B proteins (i.e., about 280,000) in the samples:

- whole brain tissue homogenates of different species (pig, tur, rabbit, rat, mouse)

microtubules prepared from these tissues by repeated cycles of polymerization and depolymerization. The hallmark of MAP is the co-polymerization with tubulin to form microtubules

- thermostable microtubule fractions. Of the microtubule building components, only MPA-2 proteins are resistant to heat denaturation.

There was no binding to tubulib proteins and other MAP f-proteins,

MAP-1 proteins).

The association of the antigenic determinant recognized by the MA-01 antibody with microtubules has been demonstrated in indirect immunofluorescence in cells of different species and tissue origins. MA-01 bound only to microtubule structures (interphase, mitotic microtubules) and no binding to other cytoskeletal structures was observed.

The MA-01 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 MA-01 hybridoma is specific for neuronal MAP 2A and MAP 2B proteins and does not need to be rid of ballast antibodies.

Example

In order to expand hybridoma cells in vivo, 6x10 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) 14 days prior to transfer of the hybridoma cells. After 13 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the produced ascites fluid collected. A total of 3.2 ml of ascites fluid containing 10 mg / ml immunoglobin was obtained. The antibody reacted with the specific antigen in an enzyme-linked immunoassay (using a horseradish peroxidase labeled porcine anti-mouse antibody) up to a 1:10 dilution of ⁴ . Using the indirect immunofluorescence technique, the thus obtained antibody was imaged by the microtubular complex in cells of the mouse fibroblastoid line C 1 H origin (up to 1:10 dilution).

MA-01 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 (called 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 with bovine serum (MA-01 hybridoma). Bioveta, Ivanovice na Hané, 10%). The hybridoma is cultured at 37 ° C. The mean generation time is 13.6 h and 6 months for construction, the modal number of chromosomes was 79. The antibody produced is an IgG4 kappa class monoclonal immunoglobin directed against the epitope present on MAP 2A and MAP 2B.

The monoclonal antibody produced by the hybridoma MA-01 analogously to conventional antibodies reacts with MAP-2 proteins of a variety of species. It is therefore useful for detecting cellular cytoskeletal structures of most species containing MAP-2 proteins.

The MA-01 hybridoma can be used industrially as a source of monoclonal antibody against MAP-2 proteins in analytical or preparative methods.

Claims (1)

Mouse lymphocyte hybridoma IMG CZAS MA-01, producing a monoclonal antibody of the IgG1 kappa light chain class, against brain MPA-2 proteins.