CS270194B1 - Mouse Lymphocyte Hybrid VU 499/1 - Google Patents

Abstract

The solution concerns a mouse lymphocytic hybridoma producing a monoclonal antibody against the glycoprotein gB of the Herpes virus. simplex type 2, stored in the hybridoma collection of the SAV Institute of Virology in Bratislava under the designation VÚ 499/1. The monoclonal antibody produced by hybridoma VÚ 499/1 is suitable for diagnostic purposes in the immunofluorescence test /IF/ in the radioimmunoassay test /RIA/ and in the immunoenzymatic analysis /ELISA/ to determine the presence and amount of viral antigen in the tested material.

Description

The invention relates to a novel hybridoma, i.e. a hybrid unicellular organism, constructed by fusion of a mouse myeloma cell Sp2/O and a mouse splenic lymphoid cell, producing an antibody against the gB glycoprotein of the Herpes simplex virus type 2.

In the Herpes simplex virus, we distinguish two antigenically distinct types of Herpes simplex type 1 and type 2. Antibodies to Herpes simplex type 2 have been prepared by immunizing experimental animals, mostly rabbits, with purified or non-purified Reap virus or some isolated protein. /Forghani 8., Schmidt N., Lennette E.H.i Solid phase radioimmunoassay for identification of herpevirus hominis types 1 and 2 from clinical material. Appl. Microbiol. 28, /1974/ 661 - 667i Dreesman G.R., Watson 0.0., Courtney R.3., Adem E., Melnick O.L.: Detection of herpesvirus type-specific antibody by microsolid-phase immunometric assay; Intervirology 12,/1979/115-119. The serum of animals immunized in this way, collected after multiple doses of antigen, served as a source of so-called type-specific antibodies, which were used to detect the Herpes simplex virus type 2 antigen in basic research and in immunodiagnostic practice. Given that Herpes simplex viruses type 1 and 2 contain not only type-specific but also type-common antigenic determinants, antibodies prepared in this way against the Herpes simplex virus circulate high levels of type-specific antibodies, which complicate or completely prevent the correct determination of the type of infecting virus in diagnostic testing. Type-specific antibodies are usually removed from the sera by saturation with Herpes simplex virus type 1, which is a very demanding procedure in terms of materials and, moreover, successful only in a small number of cases. Production batches of conventional type-specific antibodies are difficult to standardize and vary in quality. Recently, monoclonal antibodies called type-specific antibodies have been successfully used for typing Herpes simplex virus type 2, which are capable of detecting infection with Herpes simplex virus type 2 in clinical material.

The above disadvantages of the previously used procedures are unavoidable if a hybridoma cell line is available producing a type-specific monoclonal antibody against the gB glycoprotein of the Herpes simplex virus type 2, which is deposited in the hybridoma collection of the Institute of Virology of the Slovak Academy of Sciences, Mlýnská dolina 1, Bratislava under the designation VÚ 499/1.

The hybrids were obtained by a method known from the professional literature (Kohler, G., Milstein, C., Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, /1975/, 495., Gerherd, W. Fusion of cells in suspension and outgrowth of hybrids in conditioned medium. Monoclonal antibodies A new dimension in Biological analyses. Kennett R.H. et al., eds. New York, Plenum Press /1980/, 370.) Hybrid cells obtained by fusing mouse myeloma* Sp2/0 cells and cells obtained from the spleen of a BALB/c mouse immunized with an extract of cells infected with the Herpes Simplex virus type 2 were cloned and after oetetation clone VÚ 499/1 was selected.

The advantage of the hybridoma is that it produces a homogeneous antibody, the so-called monoclonal antibody, which is capable of specifically reacting with the gB glycoprotein of the Herpes simplex virus type 2. The hybridoma VÚ 499/1 can be cultivated in vitro in media suitable for animal cells or in vivo in the peritoneal cavity of BALB/o mice. Antibody production can be started from preserved cross-linked cells stored in liquid nitrogen without further immunization of the animal with antigen.

Example

In order to obtain a larger amount of monoclonal antibody VÚ 499/1 by culturing hybridoma cells in vivo, 5x10 cells were applied to the peritoneal cavity of mice. For better cell attachment, the mice were premedicated with paraffin oil 15 days before the application of cells /0.5 ml intraperitoneally per 1 mouse/. After 10 days of hybridoma growth in the peritoneal cavity, the mice were killed and the produced ascitic fluid was collected. This procedure can obtain an average of about 7 ml of ascitic fluid circulating 8 mg/ml of antibody. The ascitic fluid containing the hybridoma product VÚ 499/1 showed specific binding to Herpes simplex virus type 2 in radioimmunoassay /RIA/, in immunoenzymatic analysis /ELISA/ and

CS 270 194 Bl in the immunofluorescence test /IF/. The binding of the monoclonal antibody to the glycoprotein gB of the Herpes simplex virus type 2 was confirmed by the method of radioimmunoprecipitation and electrophoresis in polyacrylamide gel.

Hybridoma cells VÚ 499/1 grow in vitro as a semi-suspension culture. They have a spherical shape and size characteristic of myeloma cells. They contain fused cell nuclei and are aneuploid. Hybridoma cells VÚ 499/1 have an ultrastructural image of typical myeloma cells, where the predominant organelles are free and membrane-bound polyribosomes. The basic culture medium is Dulbecco's modification of Eagle's minimal essential medium (Dulbecco, R., Freeman, G., Virology 8 /1959/,' 396). This medium, referred to as DMEM, is supplemented with gentamycin and inactivated precolostral calf serum (10%, Bioveta, Ivanovice na Hané) before hybridoma cultivation. The hybridoma is cultured at 37 °C in an atmosphere of 5% C0 ₂ . Its generation time is approximately 24 h. The antibody produced is a monoclonal immunoglobulin of the IgG 1 subclass.

Hybridoma VÚ 499/1 can be used as a source of antibody to glycoprotein gB of Herpes simplex virus type 2, which can be used as qualitative evidence of the presence of Herpes simplex virus type 2 in the examined material, for quantitative determination of the amount of infecting virus or glycoprotein gB, in evaluating the epidemiological situation, for purification of glycoprotein gB and as a source of antibody of a single subclass (IgG 1) for the preparation of antisera specific for the above subclass.

Claims (1)

Mouse lymphocyte hybridoma VU IgG 1 against the gB glycoprotein of the virus 499/1, producing a monoclonal antibody of the subclass Herpes simplex type 2.