CS270543B1 - Mouse lymphocyte hybridoma vu 2-l/5 - Google Patents

Abstract

The solution relates to murine lymphocyte antibody-producing hybridoma IgG 1 subclasses against virus nucleoprotein Influenza A. Hybridoma Stored · v Collection of Hybridomas' Virological Institute SAS in Bratislava under the designation VU 2-L / 5.

Description

The invention relates to a mouse lymphocyte hybridoma, i. a hybrid single-cell organism constructed by a cell fusion of a mouse myeloma line Sp2 / 0 and a mouse elesin 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 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, is eluted as a source of antibodies, used primarily for quantitative or qualitative analysis of the antigen - influenza virus - in research and immunodiagnostic practice.

This procedure, acquired by 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, the organism produces, in addition to antibodies, a flow to the desired antigen and also antibodies to the antisera 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 need to be prepared? pure immunizing antigen and 3 other antigens to elicit ballast antibodies against non-reading.

The above-mentioned shortcomings of the above-mentioned and used procedure are discarded if a hybrid producing a monoclonal antibody against the nucleoprotein of influenza A virus, stored in the hybridoma collection of the Institute of Virology in Bratislava, Mlýnská dolina 1, under the designation VU 2-L / 5, is available. '

Said hybrid 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), 495: Gerhard. W .; Fueion of cells in suspension end outgrowth of hybrids in conditioned; _{medium.In: M o} noclonal antibodies.Hybrids: A New Dimension in Biological Analyzes.RH Kennet, T. 3. McKearn, KB Bechtol, eds., New York, Plenum Prese (1980), 370.) by cloning a set of hybrid cells after fusion! mouse myeloma line Sp2 / 0 and cells harvested from the iron of BALB / c mouse. immunized with influenza virus A / Singapore / 6/86 (HlN1). '

An advantage of the hybridon of the invention is that it produces a homogeneous antibody, the so-called a monoclonal antibody that is specifically reactive echopná a nucleoprotein of influenza virus type A hybridoma VU L-2/5 can be cultured in vitro in suitable grips for animal cells, or in vivo _in the peritoneal cavity of the mouse strain BALB / c. From cans stored in liquid nitrogen, antibody production can be initiated without further immunization with antigen. .

Example

To propagate hybridoma cells in vivo, 5x10 6 cells were injected into the peritoneal cavity of a mouse. To better adhere the applied cells, the mouse was premedicated with paraffin oil (0.5 ml intraperitoneally) 15 days before transfer of the hybridoma cells. After 20 days of hybridoma growth in the peritoneal cavity, the mouse was sacrificed and the ascitic fluid produced was collected. A total of 5 ml of ascitic fluid containing 7 mg / ml of immunoglobulin was obtained. The ascitic fluid enriching the product of the VU 2-L / 5 hybridoma showed specific binding to influenza A viruses (e.g. subtype H1, H2, H3) but not to type B in a radioimmunoassay assay and bound to influenza virus nucleoprotein in a radioimmunoprecipitation assay. A.

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 nuclei, they are aneuploid. VU 2-L / 5 hybridoma cells have an ultrastructural image of typical myeloma cells, where the right organelle is free and membrane-bound polyribosomes. The basic culture medium is Oulbecco 's modification of Eagle' s minimal essential medium (Dulbecco 's.

CS 270 543 B1 where the predominant organelles 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 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 IgG1 subclass.

The VU 2-L / 5 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 examined 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) in the preparation of sera specific for said subclass.

Claims (2)

PREOMET OF THE INVENTION Mouse lymphocyte hybrids VU 2-L / 5, producing an IgG 1 subclonal monoclonal antibody against influenza A virus nucleoprotein.