CS272888B1 - Mouse lymphocyte hybridome blvgp 51 ueo-25 e11/2 - 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-25E11/2. 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

(57) The invention relates to a murine lymphocyte hybridoma producing a neutralizing monoclonal antibody against the envelope glycoprotein gp51 of the bovine leukemia virus deposited in the hybridoma collection of the Department of Molecular Virology of the Institute of Experimental Oncology of SAS under designation UE0-25E11 / 2. The monoclonal antibody of this hybridoma itself is particularly suitable for use in the manufacture of immunodiagnostic kits for the detection of bovine leukemia-infected cattle and for the detection of a viral antigen. It is also suitable for preparative as immunofinite purification of viral glycoprotein, for the preparation of an anti-idiotypic antibody for vaccination purposes and for virus neutralization.

tn

-g i

81 272888 81

The invention relates to a novel murine lymphocyte hybridoma BLVgp51UE0-25E11 / 2, i. hybrid cell line, which was constructed by fusing mouse myeloma cells. the Sp2 / 0 line and mouse lymphoma cells producing antibodies directed against 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 diagnosing bovine leukemia.

To date, antibodies against BLV antigens have been produced by purified gp51 being repeatedly injected into the production animals, most commonly rabbits. The serum of the immunized animals collected after a period of antigen treatment 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 sera of the immunized animals, the septrum of which is different and unrepeatable in each animal. The serum thus obtained also contains antibodies to the impurities of the antigen preparation, which must then be removed by saturation. For these reasons, production batches vary widely and the quality of the polyclonal antibodies obtained is not standard.

The aforementioned drawbacks of the above-mentioned conventional immunization process will be eliminated when we have a hybridoma producing a monoclonal antibody against the envelope glycoprotein BLV, which is stored in the hybridoma collection of the Department of Molecular Virology, Institute of Experimental Oncology of the Slovak Academy of Sciences in Bratislava, ul. CSL. Army 21, under the designation UE0-25E11 / 2.

Said hybridoma was obtained essentially by a method known in the art as hybridoma technology (Kohler, G., Milstein, C .: Continous cultures of fused cell secreting antibody of predefined 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 hybridoma prepared was modified by us (Orlík, Altaner No.: Modifications of hybridoma technology yielding monoclonal anti-producing cells, J. Immunol. Methods, 115, 55-59 (1988).

An advantage of the hybridoma is that it produces homogeneous monoclonal antibodies that react with the BLV envelope glycoprotein. The hybridoma BLVgp51-25E11 / 2, can be cultured in vitro in animal cell media while being 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 without further antigen can be initiated. The antibody produced by clone 8LVgp51-25E11 / 2 reacts specifically with the BLV envelope glycoprotein determinant and is neutralizing in nature.

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 growth of hybridoma cells in the peritoneal cavity, the mouse was sacrificed and unproduced ascites fluid 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 glycoprotein gp51-BLV and with the disintegrated virus in the radioprecipitation assay and the radioimmunoassay. Immunoglobulin was isolated from ascites fluid by chromatography on a Protein A-Sepharose clone 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 i

'ί

CS 272880 B1 ° C in CO ₂ thermostate, its standard generation time is approximately 24 hours. The antibody produced is a monoclonal immunoglobulin of IgG2b subclass.

The BLVgp51UED-25E11 / 2 hybridoma can be industrially used 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 in viral glycoprotein; virus antigen in infected animals, prior to neutralization of BLV.

Claims (1)

OBJECT OF THE INVENTION Mouse lymphocyte hybridoma 8LVgp51UE0-25Ε11 / 2 producing a neutralizing monoclonal antibody of IgG2b subclass against bovine leukemia virus gp51 envelope glycoprotein.