CS267391B1 - Mouse lymphocytes hybridoma VÚIFN G8 - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody against human leukocyte interferon gamma, stored in the hybridoma collection of the Institute of Virology of the Slovak Academy of Sciences in Bratislava under the designation VÚ IFN G8. The monoclonal antibody of the hybridoma VÚ IFN G8 is suitable for determining the presence of interferon gamma based on the inhibition of its biological activity in interferon-neutralization tests. The monoclonal antibody is also suitable for the preparation of sera specific to mouse immunoglobulin of the IgGl subclass.

Description

(57) The present invention relates to a murine lymphocyte hybridoma producing an antibody to human leukocyte interferon gamma deposited in the hybridoma collection of the Institute of Virology of SAS in Bratislava under the designation of IFI G8. The monoclonal antibody hybridoma VU IFN G8 is useful for determining the presence of interferon gamma by inhibiting its biological activity in interferon-neutralization assays. The monoclonal antibody is also suitable for the preparation of sera specific for mouse immunoglobulin IgG1 subclass. EN 267391 B1 EN 267 391 B1 1

The present invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by cellular fusion of a mouse myeloma lineage NS0 and a mouse spleen lymphoid cell producing an antibody against the human gamma interferon.

To date, interferon gamma antibodies have been produced such that the interferon is repeated as an antigen to experimental animals, most often to rabbits or sheep. The serum of the animals so immunized, taken after a certain period of antigen uptake, serves as a source of antibodies used primarily for quantitative or qualitative analysis of the interferon gamma antigen in research and immunodiagnostic practice. This process, called conventional immunization, has several drawbacks. There is a heterogeneous mixture of antibodies in the serum of immunized animals, the spectrum of which is unique and unrepeatable in each individual organism. As a rule, in addition to antibodies to the desired antigen, the organism will produce antibodies to the antigen preparation impurities that need to be removed from the sera by saturation. Consequently, production batches of conventional sera are difficult to standardize and are in a wide range of quality. To produce each batch, a clean immunization antigen and other antigens need to be prepared to saturate the ballast antibodies against impurities.

The aforementioned drawbacks of the aforementioned and hitherto used process are omitted when a hybridoma producing a monoclonal antibody to human interferon gamma deposited in the hybridology collection of the Institute of Virology of SAS in Bratislava, Mlýnská dolina 1, under the designation VÚ 1FN G8, is available.

Said hybridoma was obtained by a method known from the literature (Kóhler, G., Milstein, C .: Continuous cultures of fused cells secreting antibody predefined specificity. Nature, 256, / 1975 /, 495; Gerhard, W .: Fusion of cells in suspension and outgrowth of hybrids in conditioned medium Hybrids: A New Dimension in Biological Analysis RH Kennet, TJ, McKearn, B. B. Bechtol, eds., New York, Plenum Press, / 1980/370 ) by cloning a set of hybrid cells after fusion of mouse myeloma line NSO and spleen cells of BALB / c mice immunized with human leukocyte interferon gamma preparation. The advantage of a hybridoma is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with interferon gamma. The V1 IFN G8 hybridoma can be cultured in vitro in media suitable for animal cells, or in vivo in the peritoneal cavity of BALB / c mice. From the cans stored in liquid nitrogen, antibody production can be initiated without further antigen immunization. 2 neural cavities of mice. For better attachment of the applied cells, the mice were pre-treated with paraffin oil 10.5 ml intraperitoneally 15 days prior to hybridoma cell transfer. After 20 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the produced ascites fluid collected. A total of 7 ml of ascitic fluid was obtained which contained 8 mg / ml of immunoglobulin. The ascitic fluid containing the hybridoma product of the IFN G8 hybridoma specifically inhibited the antiviral and antiproliferative activity of human interferon gamma.

In vitro growths of hybridomas as a semi-suspension culture. They are shaped (round) and the size characteristic of myeloma cells, containing fused cell nuclei, is aneuploid. IFN G8 hybridoma cells have an ultrastructural image of typical myeloma cells, where free and membrane-bound polyribosomes are the predominant organelle. The basic culture medium is Dulbecco's modification of Eagle's minimal essential medium (Dulbecco, R. and Freeeman, G., Virology 8/1959 /, 396). This medium, referred to as DMEM, is supplemented with gentamycin and inactivated horse serum (10%, Elan clone, Bratislava) to cultivate the hybridoma. The hybridoma is cultured at 37 ° C - its mean generation time is about 24 hours. The antibody produced is a monoclonal immunoglobulin of the IgG1 subclass.

The VU IFN G8 hybrid may be industrially used as a source of anti-human leukocyte interferon gamma antibody in quantitative or qualitative evaluation of the presence of interferon gamma in the investigated material, or as a single subclass (IgG1) antibody source for the production of said subclass specific sera.

Claims (1)

OBJECT OF THE INVENTION Murine Lymphocyte Hybridoma VU IFN G8, producing monoclonal antibody IgG1 subclass against human interferon gamma. Example For the purpose of propagating hybridoma cells in vivo, 5 x 10 6 cells were injected into peritoneal cells.