CS270542B1 - VU CF2 lymphocyte hybridoma mouse - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma producing an antibody of the IgG 1 subclass κ against the light chain of the hemeglutinin of influenza A virus. The hybridoma is deposited in the hybridoma collection of the Institute of Virology of the Slovak Academy of Sciences in Bratislava under the designation VU CF2.

Description

The invention relates to a murine lymphocyte hybridoma, i. a hybrid nuclear cell organism constructed by a cell fusion of a mouse myeloma line Sp2 / 0 and a mouse spleen cell producing antibodies against the light chain (HA) of hemagglutinin influenza A virus subtype H3.

Antibodies to influenza virus are hitherto prepared in such a way that the virus resp. its isolated proteins are repeatedly injected as an antigen into experimental animals, most often rabbits, mice, or ferrets. Serum from such immunized animals, collected after a certain period of antigen exposure, serves as a source of antibodies, used primarily for quantitative or qualitative analysis of the antigen - influenza virus - in research and immunodiagnostic practice.

This procedure, called conventional immunization, has several disadvantages. The serum of immunized animals contains 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 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 other antigens must be prepared to saturate the ballast antibodies against impurities.

The above-mentioned shortcomings of the above-green and obtained procedure are eliminated if a hybridoma producing a monoclonal antibody against the X-chain hemagglutinin of influenza A virus subtype H3, stored in the hybridoma collection of the Institute of Virology in Bratislava, Mlýnská dolina 1, under the designation VU CF2, is available.

Said hybridoma was obtained by a method known from the Kohler literature. G., Milstein. C .: Continuous cultures of fused cello secreting antibody of predefined specificity. Nature 256. (1975Ú, 496: Gerhard. W .: Fusion of cells in suspension and outgrowth of hybrids in conditioned medium. In: Monoclonal antibodies. Hybrids: A New Dimension in Biological Analyzes, RH Ksnnet, T. 3. McKeam, KB Bschtol, eds. influenza A / Dunedin / 4/73 (H3N2).

The advantage of the hybridoma according to the invention is that it produces a homogeneous antibody, the so-called a monoclonal antibody that is capable of specifically reacting with homagglutinin of influenza A virus of subtype H3. The VU CF2 hybrid can be cultured in vitro in media suitable for animal cells, or in vivo in the paritoneal cavity of a BALB / c mouse. From cans stored in liquid nitrogen, antibody production can be initiated without further immunization with antigen.

Example

To multiply hybridoma cells in vivo, 5x10 ⁴ cells were applied to the peritoneal cavity of the mouse. 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 sacrificed and the ascitic fluid produced was collected. A total of 7 ml of ascitic fluid containing 7 mg / ml immunoglobulin was obtained. The ascitic fluid containing the VU CF2 hybridoma product showed specific binding to influenza A virus of subtype H3, but not to other subtypes, to isolated hemagglutinin of subtype H3 and to isolated X-chain (HA) hemagglutinin of subtype H3 in a radioimmunoassay. It bound to the light chain (HA) of hemagglutinin in a radioimmunoprecipitation assay.

In vitro hybridoma cell growth as a semi-succulent culture. May's shape (gul'atý) and ver

CS 270 542 Bacteria characteristic of myeloma cells, contain fused cell nuclei, eu aneuploid. VU CF 2 hybridoma cells have an ultrastructural image of typical myeloma cells, where the predominant organelle eu are free and membrane-bound polyribosomes. The basic culture medium is Oulbecco's modification of Eagle's minimal essential medium (Dulbecco, R. and Freeman. G., Virology 8 (1959), 396). This medium, designated DMEM, is supplemented with gentamicin and inactivated precolethral er (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 igG1 subclass.

The VU CF 2 ea hybridoma can be used industrially as a source of antibody against hemagglutinin of influenza A virus subtype H3 in analytical, preparative or diagnostic methods, namely:

X 1) in the quantitative and qualitative evaluation of the presence of influenza A virus subtype H3 haemagglutinin in the investigated material! (eg clinical specimens).

, 2) in the evaluation of the epidemiological evaluation,

3) in the purification of H3 influenza A eubtype haemagglutinin,

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

Claims (1)

SUBJECT OF FOUNDING Wash lymphocyte hybridoma VU CF2, producing monoclonal igG 1 against the light chain hemagglutinin of influenza A virus eubtype antibody subclass H3.