CS270537B1 - Mouse lymphocyte hybridoma vu 144/1-2 - Google Patents

Abstract

Solution ge. mouse lymphocytic monoclonal hybridoma anti-glycoprotein antibody Herpes simplex virus type 1 and type 2 gB stored in the Virologic Nybridom Collection of the Slovak Academy of Sciences in Bratislava VU 144 / 1-2. Monolithial antibody produced by hybridoma VU 144 / 1- -2 is suitable for diagnostic purposes in immunofluorescence assay (IF), in a radioimmunoassay Assay (RIA), Immunoenzymatic analysis / ELISA / e in radioimmunoprecipient test, to determine the presence and amount of virus antigen in the test material. Oe suitable also for immunoaffinity glycoprotein purification gB from infected extracts cells.

Description

The invention relates to a novel hybridoma, i. a hybrid single cell organism, engineered by fusing a mouse Sp2 / 0 myeloma cell and a mouse alezine lymphoid cell to produce an antibody to the Herpes simplex virus type 1 and type 2 gB glycoprotein.

Antibodies to Herpes simplex virus were prepared by repeatedly purifying the virus, purified or unpurified, reap, its isolated proteins, into experimental animals, most preferably rabbits, mice or guinea pigs. Serum of such immunized animals, collected after several doses of antigen, was used as a source of antibodies used mainly for qualitative antigen detection - Herpes simplex virus - in basic research and immunodiagnostic practice (Kalírno KOK, Marttila RO, Granfors K., Viljanen MK: Solid-phase radioimmunoassay of human immunoglobulin M and immunoglobulin G antibodies against Herpes simplex virus type 1 capsid, envelope and excreted antigens (Infect. Immun. 15/1972), 883-889: Leese 3., Hana L., Metis 3 .: Reactions of immune sera against the nuclaocapsid. envelope and whole Herpes simplex virus type 1. Acta virol. 20 (1976), 48-52). This procedure, called conventional immunization, has several disadvantages. The serum of immunized animals contains a heterogeneous mixture of antibodies, the spectrum of which is different and unrepeatable in each organism. In addition to antibodies to the desired antigen, the organism produces antibodies to the cellular impurities present in the antigen. These antibodies need to be removed from the sera by saturation. Production batches of conventional antisera are difficult to standardize! and tend to be in a wide range of quality. For the production of a quality batch of aero, a sufficient amount of pure immunizing antigen must be prepared, which is especially the case with the Herpes simplex virus, which requires the material and technical equipment of the laboratory. Recently, they are increasingly being used to diagnose herpes infections! monoclonal antibodies, so-called species that are able to react with both types of Harpsa simplex virus.

The above-mentioned disadvantages of the previously used approaches and the occurrence of a hybridoma cell line producing a type-apolar monoclonal antibody against the glycoprotein gB of the Herpee simplex virus type 1 and type 2, which is stored in the hybridoma collection of the Institute of Virology of the Slovak Academy of Sciences, Mlýnská dolina 1, Bratislava under the designation / 1-2,

Said hybridoma was obtained in a manner known from the literature (Kohler, G., Milstein, C .: Continuous cultures of fused cella secreting antibody of predefined specificity. Nature, 256 (1976), 495., Gerhard, W .: Fusion of cells in suspension and outgrowth of hybrida in conditioned medium Monoclonal antibodies: A new dimension in Biological analyzes Kennett RH a apol., eda. New York, Plenum Prwss (1980), 370). Hybrid cells obtained after fusion! mouse myeloma Sp2 / 0 cells and cells obtained from the spleen of BALB / c mice immunized with a mixture of purified Herpee simplex viruses type 1 and type 2 were cloned and, after testing, clone VU 159 / 1-9 was selected.

The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called a monoclonal antibody capable of specifically responding to the Herpes simplex type 1 and type 2 gB glycoprotein. The VU 144 / 1-2 hybridoma can be cultured in vitro in media suitable for animal cells or in vivo in the peritoneal cavity of a BALB / c mouse. From canned frozen cells stored in liquid nitrogen, antibody production can be initiated without SalSej immunization with antigen. ‘

Example:

In order to obtain a larger amount of monoclonal antibody VU 144 / 1-2 by culturing hybridoma cells in vivo, 5x10 ⁶ cells aa were applied to the peritoneal cavity of the mouse. For better cell attachment, the mouse was premedicated with paraffin oil (0.5 ml intraperitoneally per 1 dish) 15 days before the application of the cells. By this procedure, an average of about 7 ml of ascitic fluid containing 8 m 2 / ml of antibody can be obtained. The ascitic fluid surrounding the VU 144 / 1-2 hybridoma product showed spasm

CS 270 537 Specific binding to Herpes simplex virus type 1 e type 2B in a radioimmunoassay (RIA) and in an immunoenzymatic analysis (ELISA). The binding of the monoclonal antibody to the gB glycoprotein of Herpes simplex virus type 1 and type 2 was determined by radioimmunoprecipitation and polyacrylamide gel electrophoresis ea.

VÚ 144 / 1-2 hybridoma cells grown in vitro as a semi-suspension culture. They have the round shape and size characteristic of breast myeloma cells. They fuse the fused cell nuclei _from being aneuploid. VÚ 144 / 1-2 hybridoma cells have an ultrastructural image of typical myeloma cells, where the predominant organelle are free and membrane-bound polyribosomes. The basic culture medium is Oulbecco's modification of Eagle's minimal essential medium (Dulbecco, R., Freeman, 6, Virology 8 (1959) .396). This medium, designated OMEM, is supplemented with gentamicin and inactivated precolostral calf serum (10%, Bioveta, Ivanovice na Hané) for hybridoma culture. The hybridoma is cultured at 37 ° C in a 5% CO 2 atmosphere. What the generation time is approximately 24 hours. The antibody produced is a monoclonal immunoglobulin of the IgG1 subclass.

and * 'A

The VU 144 / 1-2 hybridoma can be used as a source of antibody to the Herpes simplex virus type 1 e type 2 glycoprotein antibody, which can be used to detect the presence of Herpes simplex virus type 1 and type 2 virus in the test material, to quantify the amount of infectious virus resp. glycoprotein gB in the evaluation of epidemiological elution, for the purification of glycoprotein gB from extracts of infected cells by immunoaffinity chromatography and as a source of a single subclass antibody (IgG1) for the preparation of antisera specific for said subclass.

Claims (1)

OBJECT OF THE INVENTION MySi lymphocyte hybridoma VU 144 / 1-2, producing a monoclonal antibody of the IgG1 subclass against the gB glycoprotein of Herpes simplex virus type 1 and type 2.