CS270543B1 - Mice lymphocyte hybridoma VU 2-L / 5 - Google Patents

Abstract

The solution concerns a mouse lymphocyte hybridoma, producing an antibody, subclass IgG 1, against the nucleoprotein of the influenza A virus. The hybridoma is stored in the hybridoma collection of the SAV Institute of Virology in Bratislava under the designation VU 2-L/5.

Description

CS 270 543 B1 1

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

Antibodies to influenza virus proteins are prepared in such a way that the virus, respectively. its isolated proteins are repeatedly injected as antigen to experimental animals, most commonly of rabbit, mice, or ferrets. The serum of the animals so immunized, taken after a certain period of antigen challenge, serves as a source of counterparts used primarily for the quantitative or qualitative analysis of the antigen - influenza virus - in research and practice.

This process, acquired by conventional immunization, has several drawbacks. There is a heterogeneous mixture of antibodies in the serum of the immunized animals, 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 will produce antibodies to the anti-gene preparation impurities that are required to be removed by sera. Consequently, batches of conventional sera can be heavily standardized and are in a wide range of quality. The production of each batch requires the preparation of a clean immunization antigen and more antigens to saturate the ballasts for impurities.

The above mentioned drawbacks of the highly misaligned and disposed of the used waste when available! a hybridoma, producing a monoclonal antibody against the influenza A virus nucleoprotein deposited in the hybridoma collection of the Virological Institute of SAS in Bratislava, Mlynskádolina 1, under the designation VU 2-L / 5.

Said hybridoma was obtained by a method known in the art (Kohler, G.,

Milstein, C.,: Continuous cultures of fused cella secreting antibody of predefined spicy. Nature 256, (1975), 495: Gerhard. W .; Fuslon of cells in suspension and outgrowthof hybrlds in conditioned medium. In: M0noclonal antibodies. Hybrids: A New Olmensionin Biological Analyzes. RH Kennet, T. 3. McKearn, KB Bechtol, eds., New York, Plenum Press (1980), 370.) cloning a set of hybrid cells after fusion of mouse myeloma sp2 / 0 and cells obtained from mySi spleen strain BALB / c. immunized with influenza virus A / Singapora / 5/86 (H1N1). An advantage of the hybridoma of the invention is that it produces a homogeneous antibody, tzV. a monoclonal antibody that is capable of specifically reacting with a type A influenza virus nucleoprotein. VU 2-L / 5 hybridoma can be cultured in vitro in media suitable for animal cells or in the peritoneal cavity of the mySi BALB / c strain. From the cans stored in liquid nitrogen, antibody production can be started without further anti-genoa immunization. Example To propagate hybridoma cells in vivo, 5x10 6 cells of the doperitoneal cavity, the mouse, were applied. For better attachment of the applied cells, myS was prewashed with paraffin oil (0.5 ml intraperitoneally) for 15 days prior to hybridoma cell transfer.

After 20 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the produced ascetic fluid collected. A total of 5 ml of ascitic fluid containing 7 mg / ml of immunoglobulin was obtained. The ascites fluid containing the hybridoma VU 2-L / 5 product exhibited a specific binding to the influenza A viruses (e.g., subtype H1, H2, H3) but not to the B type in the radioimmunoassay, and bound to the nucleo-protein of the virus in the radioimmunoassay. influenza A.

In vitro growth of a hybridoma cell as a semi-suspension culture. Their shape (guTaty) and the size characteristic of myeloma cells contain fused cell nuclei, they are aneuvid. VU 2-L / 5 hybridoma cells have an ultrastructural image of typical myeloma cells, where the matching organelle is free and membrane-bound polyribosomes. The basic culture medium is Oulbecco's modification of Eagle's minimal essential medium (Dulbecco.

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.