CS272886B1 - Mouse lymphocyte hybridome blvgp 51 ueo-6a12/a7 - 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-6A12/A7. 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, preparation of the anti-idiotype antibodies for vaccination purposes and neutralisation of the virus.

Description

The invention relates to a novel murine lymphocyte hybridoma BLVgp51UE0-6A12 / A7 i.e. hybridoma cell line, which was constructed by fusing cells of the murine myeloma line Sp2 / 0 and murine lymphoid cells producing antibodies directed to the envelope glycoprotein gp51 of bovine leukemia virus (BLV). 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 BLV antigens have been produced by purified gp51 being injected repeatedly into production animals, most commonly rabbits. The serum of animals thus immunized, collected at 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, the sera of the immunized animals contain a heterogeneous mixture of antibodies, the spectrum of which is distinct and unique in each animal. The serum thus obtained also contains antibodies to the antigen preparation impurities, which must then be removed by saturation. For these reasons, the production batch differs considerably and the quality of the polyclonal antibodies obtained is not standard.

The above drawbacks of the above-mentioned conventional immunization wastes process, when available! hybridon, producing a monoclonal antibody against envelope glycoprotein BLV, which is deposited in the collection of hybridomas of the Department of Molecular Virology, Institute of Experimental Oncology of the Slovak Academy of Sciences in Bratislava, ul. CAA. Army 21, under the designation UE0-6A12 / A7.

Said hybridoma was obtained essentially by a method known in the literature as hybridoma technology (Kohler, G., Milstein, C. of Continued Cultures of Fused Cell Secreting Antibody of Prodefined Specificity, Nature 256, 495, 1975), R. H. Burdon, Ρ. H. VanKnippenberg: Laboratory techniques in biochemistry and molecular biology, Vol. 13, Monoclonal Antibody Technology, Elsevier, Amsterdam 1984). The procedure by which the hybridomas were prepared has been modified by us (Orlik, N. Altaner: Modifications of hybridoma technology yield of monoclonal antibody producing cells, J. Immunoi. Methods, 115, 55-59, 1988).

The advantage of the hybridoma is that it produces homogeneously monoclonal antibodies that react with the BLV envelope glycoprotein. The hybridoma SLVgp51-6A12 / A7 can be cultured in vitro in media suitable for animal cells while being adapted for growth in vivo in the peritoneal cavity of the 8ALB / c mouse strain. From cans that are stored in liquid nitrogen, antibody production can be initiated without additional antigen. The antibody produced by the clone BLVgp51-6A12 / A7 reacts specifically with the determinant of the envelope glycoprotein 8LV and has a neutralizing character.

Example

To expand hybridoma cells in vivo, 2-10 x 10 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 Incomplete Freud's Adjuvant to create sterile ascites that aided the growth of injected hybridoma cells. After 14 days of hybridoma cell growth in the peritoneal cavity, the mouse was sacrificed and the ascitic fluid produced was harvested sterile. A total of 8 ml of ascites fluid containing 9 mg / ml immunoglobulin was obtained. The monoclonal antibody reacted specifically with the gp51-BLV glycoprotein and the disintegrated virus in the radio-precipitation and radioimmunoassays. Immunoglobulin was isolated from the ascites fluid by Protein A-Sepharese column chromatography or other suitable method. The monoclonal antibody thus purified is suitable for coating polystyrene materials (microtiter plates, polystyrene rods) in order to prepare diagnostic kits for detecting BLV infected animals.

In vitro hybridoma cell growths as a semi-suspension culture. The basic culture medium is Dulbecco's modification of Eagle's minimum essential medium supplemented with 5 percent of horse or other tissue culture-appropriate serum. The hybridoma is cultured at

CS 272006 f11 2 ° C in C0 ₂ thermostate, its standard generation time is approximately 24 hours. Produkovaná. the antibody is a monoclonal immunoglobulin of IgG1 subclass.

The BLVgpUEO-6A12 / A7 hybridoma can be used industrially as a source of antibody to the gp51 BLV envelope glycoprotein in diagnostic methods, purification methods, and other applications.

It has particular applications: in the production 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, in anti-idiotypic antibody virus antigen in infected animals prior to BLV neutralization.

Claims (1)

OBJECT OF THE INVENTION The murine lymphocyte hybridoma BLVgp51UE0-6Al2 / A7, producing a neutralizing monoclonal antibody of IgG1 subclass against the bovine leukemia virus gp51 envelope glycoprotein.