CS259995B1 - Mouse lymphocyte hybridoma producing anti-acrosine monoclonal antibody - Google Patents

Abstract

Mouse lymphocyte hybridoma, producing an antibody against acrosin, which reacts with acrosin of pig, bull, ram and mouse, is stored in the hybridoma collection of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences under the designation MO. AKR. 4. The monoclonal antibody of hybridomas MO. AKR. 4 itself is suitable for use in radioimmunological or enzyme-immunological analysis for determining the amount of pig, bull, ram and mouse acrosin. In the immunofluorescence test, it marks an undamaged acrosome in pig, bull, ram and mouse.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by cell fusion of a myeloma mouse line and a mouse spleen lymphoid cell producing an anti-porcine acrosin antibody.

Anti-acrosin antibodies are produced by repeatedly injecting acrosin as an antigen into test animals, most commonly mice or rats. The serum of the immunized animals collected after a period of antigen treatment serves as a source of antibodies used to determine acrosin in damaged spermatozoa. 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 generates antibodies to the desired antigens as well as antibodies against the impurities of the antigen preparation. These must be removed from the sera by saturation. Batches of conventional sera are therefore difficult to standardize. For the production of each batch, it is necessary to prepare a pure immunization anti-gene and other antigens to saturate the ballast antibodies against impurities.

Yippee. a known monoclonal antibody against porcine acrosin,

AKR.l, which is deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Videnska No. 1083, under the name IMG CZAS MO. AKR 1.

Another monoclonal antibody against acrosin is produced by the new hybridoma MO AKR.4 according to the invention, which is deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská

δ. 1083, under the designation IMG CZAS MO. ACRE. 4. This hybridoma produces a monoclonal antibody against porcine, bull, ram and murine acrosin, thus reacting interspecies, similar to a conventional antibody prepared; however, it also has all the advantages of a monoclonal antibody (homogeneity, standardity, ease of preparation of large amounts of antibody). In the immunofluorescence assay / Immunochemistry in: Immunochemistry in Practice, eds A. Johnstone, R. Thorpe, p 256. 1982 /, indicates a monoclonal antibody of the MO hybridoma. ACRE. 4 acrosome of pig, bull, ram and mice in intact sperm.

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Okay

Said hybridoma was obtained by a method known in the literature / Fazekas de St. Groth, Sat, Scheidigger, 0 .: Product of Monoclonal Antibodies: Strategies and Tactics, J. Immunol. Met., 35: 1-21 (1980); Galfré, G., Howe, SG, Milstein, C., Butcher, GW, Howard, JC: Antibodies to major histocompatibility antigens produced by hybrid cell line, Nature 266: 550, 1977 / by cloning a set of hybrid cells resulting from fusion of mouse myeloma line Sp 2 / o-Ag 14 and cells obtained from the spleen of BALB / c mice immunized with acrosin.

Hybridom MO. ACRE. 4 can be cultured in vitro in animal cell media and adapted for in vivo growth in the peritoneal cavity of BALB / c mouse mice. From cans stored in liquid nitrogen, antibody production can be started without additional antigen.

example

To expand the 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 mice were treated with paraffin oil (0.5 ml intraperitoneally) 10 days prior to transfer of the hybridoma cells. After 10 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the produced ascites fluid collected. A total of 5 ml of ascites fluid was obtained which contained >

mg / ml immunoglobulin, IgM class. The monoclonal antibody contained in the ascites fluid reacts with acrosin of various species (pig, bull, ram, mouse) in the ELISA assay until dilution (10 @ -1).

MO hybridoma cells. ACRE. 4 have an ultrastructural picture of typical rayeloma cells. They grow in vitro as semi-suspension cultures. The basic culture medium is RPMI 1640 / Moore, G.E. et al. J. Am. Copper. Assoc., 199: 519, 1967 (prepared by the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences, supplemented with glutamine, HEPES, antibiotics and 10% calf serum (Bioveta, Ivanovice na Hané)). H. hybridoma is cultured at 37 ° C and 5% CO 2. The mean generation time is 17.3 hours.

Hybridom MO. ACRE. 4 can be used industrially as a source of antibody not only against porcine acrosin but also acrosin of bulls, rams and in mouse research. Using this antibody, the amount of acrosin released in damaged spermatozoa of different animal species can be determined and thus tested for various semen insemination doses after thawing and selecting suitable (fertile, undamaged) for insemination.

In the ELISA assay, the amount of acrosin is determined by the supernatant of the collected sperm from animals or thawed sperm containing the released acrosin is dropped into Terasaki plates (Dalecin). The monoclonal antibody MO is added to the instilled antigen-acrosin. ACRE. After incubation of the antigen with the monoclonal antibody, a peroxidase-labeled porcine anti-mouse iraunoglobulin (SwAM Px, USOL, Prague) is added, after the addition of ortho-phenyldiamine (peroxidase substrate), a color reaction is obtained which can be visually read.

Claims (1)

Mouse Lymphocytic Hybridoma IMG CZAS MO. ACRE. 4, characterized in that it produces a monoclonal anti-acrosin antibody that reacts with acrosin of pig, bull, ram and mice. Uzhgorod Manufacturing-Patent Company ”, Gagarina 101, Uzhgorod H · 2330, Price £ 2.40