CS270544B1 - Mouse lymphocyte hybridoma vu iih3 - Google Patents

Abstract

The present invention relates to murine lymphocyte antibody-producing hybridoma IgG 1 subclasses of qukleoprotein virus Influenza The hybridoma is stored in collection of hybridomas of the Vlrological Institute SAS in Bratislava under the designation VU IIH3.

Description

The invention relates to a murine lymphocyte hybridoma, i. a hybrid single-cell organism constructed by a cell fusion of a mouse myeloma line Sp2 / 0 and a mouse spleen cell producing an antibody against influenza A virus.

Antibodies to influenza virus proteins have so far been prepared in such a way that the virus or. its isolated ee proteins are repeatedly injected as an antigen into experimental animals, most commonly rabbits, mice, or ferrets. Serum from such immunized animals, collected after a certain period of antigen challenge, is used as a source of antibodies, used primarily for quantitative or qualitative analysis of the antigen - influenza virus - in research and immunodiagnoetic practice.

This procedure, not called conventional immunization, has several disadvantages. In the serum of immunized animals, ea finds a heterogeneous mixture of antibodies, the spectrum of which is spicy and unrepeatable in each individual organism. As a rule, in addition to antibodies to the desired antigen, the organism also produces antibodies to the impurities of the antigen preparation, which need to be removed from the sera by saturation. Production batches of conventional sera are therefore difficult to standardize and tend to be in a wide range of quality. For the production of each batch, pure immunizing antigen and Salsi antigens must be prepared to saturate the ballast antibodies against impurities.

The above-mentioned shortcomings of the above-mentioned and obtained procedure will be eliminated if a hybridoma producing a monoclonal antibody against the influenza A virus nucleoprotein, stored in the collection of hybridomas of the Virological Institute of the Slovak Academy of Sciences in Bratialev, Mlýnská dolina 1, under the designation VU IIH3, is available.

Said hybridoma was obtained by a method known from the literature (Kohler, G., Milstein, C .: Continuous cultures of fused cells secreting antibody of predefined specificity, Nature 256 (1975), 486t Gerhard. W .: Fusion of cells in suspension and outgrowth of hybrids in conditioned medlue Int Intoclonal antibodies Hybrids: A New Dimension in Biological Analysis RH Kennet, T. Mc McKearn, KB Bechtol, eds., New York Plenum Preee (I960), 370.) by cloning a set of hybrid cells after the merger! mouse myeloma line Sp2 / 0 and cells obtained from the spleen of BALB / c mouse strain immunized with influenza A / Dunedin / 4/73 (H3N2) virus. ;

The advantage of the hybridoma according to the invention is that it produces a homogeneous antibody, the so-called a monoclonal antibody capable of specifically responding to nucleoprotein influenza A virus. The VU IIH3 hybridoma can be cultured in vitro in pups suitable for animal cells, or in vivo in the peritoneal cavity of a BALB / c mouse strain. From cans stored in liquid nitrogen, antibody production can be initiated without further immunization with the entigen.

Example * 6

To propagate hybridoma cells in vivo, ee applied 5x10 6 cells to the peritoneal cavity of mice. To better adhere the applied cells, the mouse was premedicated with paraffin oil (0.5 ml intraperitoneally) 15 days before transferring the hybridoma cells. After 20 days of growth of the hybridoma in the peritoneal cavity, the mouse was killed and the ascitic fluid produced was collected. A total of 9 ml of ascitic fluid containing 6 mg / ral of immunoglobulin was obtained. The aecithic fluid containing the VU IIH3 hybridoma product showed specific binding to influenza A viruses (e.g., subtype H1, H2, H3) but not type B in a radioimmunoassay, and in the radioimmunoprecipitation ea and bound to influenza A nucleoprotein.

In vitro hybridoma cell growth as a semi-suspension culture. They have the shape (round) and size characteristic of myeloma cells, they contain fused cell foods, they are aneuploid. VU IIH3 hybridoma cells have an ultrastructural image of typical myeloma cells, where the predominant organelle is free and membrane-bound polyribosomes. The basic culture medium is Oulbecco's modification of Eagle's minimal essential medium (Oulbecco. R.

CS 270 544 B1 and Freeman, G., Virology 8 (1959), 396). This medium, designated DMEM, is supplemented with gentamicin and inactivated with precolostral serum (10%, Bioveta, Ivanovice na Hané) for hybridoma culture. The hybridoma is cultured at 37 ° C - its mean generation time is approximately 24 hours. The antibody produced is a monoclonal immunoglobulin of the IgG 1 subclass.

The VU IIH3 hybridoma can be used industrially as a source of antibody against the nucleoprotein (nucleocapsid protein) of influenza A virus in analytical, preparative or diagnostic methods, namely:

1) in the quantitative and qualitative evaluation of the presence of the influenza A virus nucleoprotein in the investigated material! (eg clinical specimens), 2) when evaluating the epidemiological situation,

3) in the purification of the influenza A virus nucleoprotein,

4) as a source of a single subclass antibody (IgG 1) for the preparation of sera specific for said subclass.

Claims (1)

BEFORE THE INVENTION Mouse lymphocyte hybridoma VU IIH3, producing an IgG 1 subclonal monoclonal antibody against influenza A virus nucleoprotein. ·