CS270192B1 - The mouse lymphocyte mutants of VÚ T96 / 4 - Google Patents

Abstract

The present invention relates to a mouse lymphocyte hybridoma producing a monoclonal antibody against the glycoprotein gC of the Herpes simplex virus type 1, stored in the hybridoma collection of the Institute of Virology of the Slovak Academy of Sciences in Bratislava under the designation Vu T96/4. The monoclonal antibody produced by the hybridoma VU T96/4 is suitable for diagnostic purposes in the immunofluorescence test /IF/ in the radioimmunoanalytical test /RIA/ and in the immunoenzyme-linked assay /ELISA/ for determining the presence and amount of viral antigen in the tested material. It is also suitable for immunoaffinity purification of the glycoprotein gC from extracts of infected cells.

Description

The invention relates to a new hybrid, i.e. of a hybrid single-celled organism, constructed by the fusion of a mouse myeloma cell Sp2/0 and a mouse allesine lymphoid cell, producing an antibody to the glycoprotein gC of the Herpes simplex virus type 1.

In the Herpes simplex virus, we distinguish two antigenically distinct types, Herpes simplex type 1 and type 2. Until now, antibodies /antisera/ to the Herpes simplex type 1 virus have been prepared by immunizing experimental animals, most often rabbits, with purified or non-purified virus or selected isolated protein /Forghani B., Schmidt N,, Lennette EH t Solid phase radioimmunoassay for identification of herpesvirus hominis types 1 and 2 from clinical material.Appl. Microbiol. 28, /1974/ 661 - 667:' Dreesman GR, Watson DO,, Courtney RJ, Adam E. _t Melnick JL: Detection of herpesvirus type-specimen antibody by moloraolid-phae radioimmunometric assay: Intervirology 12. /1979/ 115 - 119 )· Serum from animals thus immunized, collected after several doses of antigen, served as a source of so-called type-specimen antibodies, which were used to detect the Herpes simplex virus type 1 antigen in basic research and in immunodiagnostic practice. Considering that Herpes simplex viruses type 1 and 2 contain not only type-specimen but also type-specific antigenic determinants, the antisera prepared in this way against Herpes simplex virus type 1 contain high levels of type-specific antibodies, which consist of resp. completely prevent! correct determination of the type of infecting virus in diagnostic testing. Type-specific antibodies are usually removed! from sera by saturation with Herpes simplex virus type 2, but the procedure is very specific to the material and is only successful in a small number of cases. Production batches of conventional type-specific antisera can be easily standardized and are in a wide range of quality. In Poland, monoclonal antibodies, so-called type-specific antibodies, which are capable of identifying infection with Herpes simplex virus type 1 in clinical material, have been successfully used for typing Herpes simplex virus type 1.

The above disadvantages of the methods used so far do not occur if a hybridoma cell line producing a type-specific monoclonal antibody directed against the glycoprotein gC of the Herpes simplex virus type 1 is available, which is deposited in the collection of hybridomas of the Virological Institute of the Slovak Academy of Sciences, Mlýnská dolina 1, Bratislava under the designation VÚ 96/4.

The above hybrids were 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.: Fusion of cells in suspension and outgrowth of hybrids in conditioned media Monoclonal antibodies: A new dimension In Biological analyses. Kennett R. H. et al., eds. New York, Plenum Prose /1980/, 370.) Hybrid cells obtained by fusion of mouse myeloid Sp2/0 cells and cells obtained from the spleen of a BALB/o mouse immunized with an extract of cells infected with Herpes simplex virus type 1, were cloned and after testing, the VÚ T96/4 clone was selected. The advantage of the hybrid is that it produces a homogeneous antibody, the so-called. monoclonal antibody, which is capable of specifically reacting with the Herpes simplex virus type 1. Hybrids of the T96/4 virus can be cultured in vitro in media suitable for biliary cells or in vivo in the nasal cavity of BALB/o mice. From preserved frozen cells stored in liquid nitrogen, antibody production can be induced without immunization of the animal with the antigen.

Example:

In order to obtain a larger amount of monoclonal antibody VU T96/4 by culturing hybrid cells in vivo, 5x10$ cells were applied to the peritoneal cavity of mice. For better cell survival, the mice were premedicated with paraffin oil (0.5 ml intraperitoneally per 1 mouse) 15 days before the application of cells. After 10 days of growth of the hybrid in the peritoneal cavity, the mice were killed and the produced ascitic fluid was collected. This procedure yielded an average of about 7 ml of ascitic fluid containing 8 mg/ml of antibody. The ascitic fluid containing the hybrid product VU 96/4 showed specific binding to Herpes simplex virus type 1 in radioimmunoassay (RIA), immunoenzyme assay (ELISA) and immunofluorescence assay (IF). By the method of immunoprecipitation and electrophoresis

CS 270 192 Bl in polyacrylamide gel aa gold binding of monoclonal antibody to glycoprotein gC of Herpes simplex virus type 1.

Hybridoma cells of the VÚ T96/4 are grown in vitro as a semi-suspension culture. They have a spherical shape and size characteristic of myeloma cells. They contain phased cell nuclei and are aneuploid. Hybridoma cells of the VÚ T96/4 have the ultrastructural appearance of typical myeloma cells, with a predominance of organelles and free and membrane-bound polyribosomes. The basic culture medium is Dulbecco's modification of Eagle's minimal essential medium (Dulbecco, R., Freeman. G., Virology JB /1959/, 396 ). This medium, referred to as DMEM, is supplemented with gentamicin and inactivated precoelomate body serum (10$, Bloveta, Ivanovice na Haní) for the culture of hybridomas. The hybrid is cultivated at 37 °C in an atmosphere of 5¼ C0 ₂ . Its generation time is approximately 24 h. The produced antibody is a monoclonal immunoglobulin of the IgG 2b subclass. ,

Hybrids of the VU T96/4 strain can be used as a source of antibodies to the glycoprotein gC of the Herpes simplex virus type 1, which can be used for qualitative detection of the presence of the Herpes simplex virus type 1 in the cultured material, for quantitative determination of the amount of infectious virus or glycoprotein gC, in the evaluation of the epidemiological situation, for purification of glycoprotein gC from extracts of infected cells using immunochromatography and as a source of antibodies of a single subclass (igC 2b) for the preparation of antibodies to the above-mentioned subclasses. , '

Claims (1)

PRBDMET V Y N A L E Z V Mouse lymphocyte hybridoma VU T96/4, producing a monoclonal antibody of the IgG 2b subclass against the glycoprotein gO of the Herpes simplex virus type 1. Corrections in printed descriptions of the invention In the printed description of the invention to the author's certificate no. 270 192 (PV 8150-88,X) the description number is incorrectly printed at the bottom left.