CS272890B1 - Mouse lymphocyte hybridome blvgp 51 ueo-4e9/32e5 - Google Patents

Abstract

The invention concerns mouse lymphocyte hybridome producing neutralising monoclonal antibody against enveloped glycoprotein gp51 of the bovine leukaemia virus, stored in the hybridome collection of the Department of Molecular Virology of the Institute of Experimental Oncology SAV under ref. number UEO-4E9/32E5. The actual monoclonal antibody of this hybridome is suitable for use for production of immunodiagnostic sets for detection of beef cattle infected with the virus of bovine leukaemia and for detection of the virus antigen. It is also suitable for the preparatory purposes such as immuno-affinity purification of the virus glycoprotein.

Description

The invention relates to a novel mouse lymphocyte hybridoma, BIiVgp51 UEO-4E9 / 32E5 i.e. a hybrid cell line constructed with a phase of murine myeloma cell line Sp2 / 0 and murine lymphoid cells producing antibodies directed against the agp 51 envelope bovine leukemia virus (BLV) envelope glycoprotein. The envelope glycoprotein is an important component of the bovine leukemia virus and is responsible for the infectivity of the virus. The finding of anti-gp-antibodies in animals is essential for the diagnosis of bovine leukemia.

To date, antibodies against ELV antigens have been produced such that purified gp51 is repeatedly injected into production animals, most commonly. rabbits. The serum of the immunized animals collected after a certain time of antigen challenge serves as a source of antibodies. This procedure has many disadvantages, for each immunization it is necessary to isolate the gp51 glycoprotein again from the virus, its purification is expensive and time consuming. In addition, there is a heterogeneous mixture of antibodies in the serum of the immunized animals, the spectrum of which is fair and non-repeatable in each animal. The serum thus obtained also contains antibodies to the impurities of the antigen preparation, which must then be removed by saturation. Of these, the batches of the manufacturing batch differ significantly and the quality of the polyclonal antibodies obtained is not standard.

The above drawbacks of the above-mentioned conventional immunization procedure are eliminated when we have at our disposal! hybridoma, producing a monoclonal antibody against envelope glycoprotein BEiV, which is deposited in the collection of hybrids of the Department of Molecular Virology, Experimental Oncology of the Slovak Academy of Sciences in Bratislava, ul. CAA. Army 21, under the designation UEO-4E9 / 32E5.

Said hybridoma was obtained essentially by a method known in the literature as hybridoma technology (Kohler, G., Milstein, C .: Continuous Cultures of Fused Cell Secreting Antibody of Prodefined Specificity, Nature 25 ', 495, 1975), H.H. Burdon, P.H. VanKhippenberg: Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Monoclonal Antibody Technology, Elsevier, Amsterdam 1984). The procedure by which the hybridomas were prepared was modified by CO. Orlík, 5. Altaner: Modifications of hybridoma technology yielding monoclonal antibody-producing cells, J. Immunol. Methods, 115,

55-59, 1988).

The advantage of the hybridoma is that it produces homogeneous monoclonal antibodies that react with the BLV envelope glycoprotein. The BLVgp51UEO-4E9 / 32E5 hybridoma can be cultured in vitro in animal cell media and is also adapted for growth in vivo in the peritoneal cavity of the BALB / c mouse strain. From cans that are stored in liquid nitrogen, antibody production can be initiated without additional antigen. The antibody produced by the BLV clone gp51-4E9 / 32E5 reacts specifically with the BLV envelope glycoprotein determinant.

Example: In order to expand the hybridoma cells in vivo, 2-10x10 6 cells were injected into the peritoneal cavity of BALB / c mice. These mice were injected 14 days prior to the peritoneal cavity with 0.5 ml of Landing or an incomplete Preud's adjuvant to create sterile ascites that aided the growth of the injected hybridoma cells. After 14 days of hybridoma cell growth in the peritoneal cavity, the mouse was sacrificed and unproduced ascites fluid was harvested sterile. A total of 8 ml of ascites fluid containing 9mg / ml immunoglobulin was obtained. The monoclonal antibody reacted specifically with the gp51-ELV glycoprotein and with the disintegrated virus and the radioprecipitation and radioimmunoassays. This purified monoclonal antibody is suitable for coating polystyrene materials (microtiter plates, polystyrene rods) to prepare diagnostic kits for the detection of ELV-infected animals.

CS 272890 EL

In vitro hybridoma cell growths as a semi-suspension culture. The basic culture medium is Dulbecco ' s modification of Eagle's minimal esenoial medium supplemented with 5 percent of horse or other tissue culture-appropriate serum. The hybrids are cultured at 37 ° C in a CO ₂ thermostate, with a standard generation time of approximately 24 hours. The antibody produced is an IgG2b monoclonal immunoglobulin.

The BLVgp51UEO-4E9 / 32E5 hybrid can be industrially used as a source of anti-gp51 BLV envelope glycoprotein antibody in diagnostic methods, purification methods, and other applications.

In particular, it has utility: in the manufacture of immunodiagnostic kits for the detection of bovine leukemia virus infection in bovine leukemia virus, in the purification of viral glycoprotein by immunoaffinity chromatography, in the evaluation of antigenic variability of viral glycoprotein as a single subclass antibody source for sera preparation;

Claims (2)

The murine lymphocyte hybridoma BLVgp51UEO-4E9 / 32E5 producing a monoclonal antibody of IgG2b subclass against the bovine leukemia virus gp51 envelope glycoprotein.