CS255844B1 - Mouse lymphocyte hybridoma producing monoclonal antibody against porcine acrosine - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody against porcine acrosin (beta-form of acrosin), stored in the hybridoma collection of the Molecular Genetics Collection of the Czechoslovak Academy of Sciences under the designation MO.AKR.l. The monoclonal antibody of the hybridoma MO.AKR.l itself is suitable for use in radioimmunological or enzyme-immunological analysis for determining the amount of porcine acrosin.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by cell fusion. mouse myeloma lines SP 2/0 - Ag 14; and mouse spleen lymphoid cells producing anti-porcine acrosin antibody.

When anti-acrosin antibodies are produced, they are made by repeatedly injecting the 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 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 antigen of interest; these must be removed from the sera by saturation. therefore, batches of conventional sera are difficult to standardize and come in a wide quality range. Pure immunization antigen and other antigens should be prepared for each batch to saturate ballast antibodies against impurities. A conventional anti-acrosin antibody often reacts with proacrosin, which is found in undamaged spermatozoa, and therefore the degree of sperm damage caused by the release of the acrosin enzyme cannot be clearly determined by such an antibody.

The above mentioned drawbacks of the above-mentioned and hitherto used procedure will be eliminated if a hybridoma producing a monoclonal antibody against porcine acrosine, deposited in the Collection of Hybridomas of the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences in Prague 4, Vídeňská No. 1,083, is available IMG CZAS MO.AKR 1.

The hybridoma was obtained by a method known in the literature (Fazekas de St. Groth, p., Schei.digger, 1990: Production of monoclonal antibodies: Strategies and tactics, J. Immunol. Met., 35: 1-21 21 1980) Galfré, G., Howe, SC, Milstein, C., Butcher, GW, Howard,

· Cloning of a set of hybrid cells resulting from the fusion of mice of the myeloma line SP 2/0-Ag 1.4 and 'cells obtained from the spleen of BALB / mouse mice c, immunized with acrosin.

The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with porcine acrosin (specifically with the beta-form of acrosin). MO.AKR 1 hybridoma can be cultured in vitro in animal cell media and is adapted for growth in vivo in the peritoneal cavity of BALB / c mouse mice. From preserves stored in liquid nitrogen, production of the antibody can be initiated from another antigen.

Example

To enhance hybridoma cells / in vivo, 1.5 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) 10 days prior to transfer of the hybridoma cells. After 16 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the produced ascites fluid was collected. A total of 5 ml of ascites fluid containing 7 mg / ml immunoglobulin was obtained. The monoclonal antibody contained in the ascites fluid reacts specifically with beta-acrosin in the ELISA assay until dilution (10). The specificity of the monoclonal antibody was further determined by electrophoretic transfer of an antigen separated by SDS PAGE (Laemmli, UK, Nature, 227: 680, 1970) to a nitrocellulose membrane and detected by an indirect enzyme-immunoassay (Towbin, H., et al., Proc, Nat'Ac. Acad. Sci., 76: 4,350, 1979). The monoclonal antibody (MO.AKR 1) reacted only with a zone corresponding to the molecular weight of beta-acrosine (m. V. 34,000).

MO.AKR 1 hybridoma cells have an ultrastructural pattern of typical myelic cells. They grow in vitro as semi-suspension cultures. The basic culture medium is RPMI 1640 (Moore, GE et al. J. Am. Med. Assoc., 199: 519, 1967) prepared by the Institute of Molecular Genetics of the Czechoslovak Academy of Sciences, supplemented with glutamine, mercaproethanol, HEPES, antibiotics and 10% veal. serum (Bioveta, Ivanovice na Hané). The hybridoma is cultured at 37 ° C and 5% CO ₂ . The mean generation time is 16.7 hours. The modal number of chromosomes is 86.

The MO.AKR 1 hybridoma can be industrially used as a source of anti-porcine acrosin antibody. Since the antibody specifically reacts with the beta-form of acrosin, this antibody can be used to determine the amount of acrosine released in damaged spermatozoa, thereby testing various semen insemination doses after thawing and selecting appropriate (fertile, undamaged) for inseraination.

Claims (1)

Mouse lymphocyte hybridoma IMG CZAS MO.AKR 1, producing a monoclonal antibody against porcine acrosin beta-form.