CS270193B1 - Mouse Lymphocytic Hybrid VU 303/4 - Google Patents

Abstract

The solution concerns a mouse lymphoitary hybridoma, producing a monoclonal antibody against glycoprotein gG of the Herpes simplex virus type 2, stored in the collection of hybridomas of the SAV Institute of Virology in Bratislava under the designation VÚ 303/4. The monoclonal antibody produced by the VÚ 303/4 hybridoma is suitable for diagnostic purposes in the immunofluorescence test (IF), in the radioimmunoanalytical test (RIA), in the immunoenzymatic analysis (ELISA) and in the radioimmunoprecipitation test, to determine the presence and amount of viral antigen in the treated material. Oe also suitable for immunoafflinite purification of glycoprotein gG from extracts of infected cells.

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 spleen lymphoid cell, producing an antibody against the gG glycoprotein of the Herpes simplex virus type 2.

In the Herpes simplex virus, we distinguish two antigenically different types, Herpes simplex type 1 and type 2. Until now, antibodies (antibodies) to the Herpes simplex type 2 virus have been prepared by immunizing experimental animals, most often rabbits, with purified or non-purified virus or some isolated protein-(Forghani B., Schmidt N., Lennette E.H.: Solid phase radioimmunoassay for identification of herpesvirus hom-inis types 1 and 2 from clinical material. Appl. Microbiol.' 28, (1974) 661-667) Dreeaman G.R., Courtney R:J., Adam E., Melnick O.L.t Detection of herpesvirus type-specific antibody by microsolidphase radioimmunometric assay) Intervirology 12, (1979) 115-119),. The serum of animals immunized in this way, collected with 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. Since Herpes simplex viruses type 1 and 2 contain not only type-specific but also type-common antigenic determinants, antibodies to Herpes simplex virus prepared in this way contain high levels of type-common antibodies, which make it difficult or even impossible to correctly determine the type of infecting virus during diagnostic testing. Type-common antibodies are usually removed from sera by saturation with Herpes simplex virus type 1, which is a very material-intensive procedure and, moreover, only successful in a small number of cases. Production batches of conventional type-specific antisera are difficult to standardize and have a wide range of quality. Recently, so-called type-specific monoclonal 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 methods used so far do not occur if a hybridoma cell line producing a type-specific monoclonal antibody against the gG glycoprotein of the Herpes simplex virus type 2 is available, 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 VÚ 303/4.

The above hybridoma 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.: 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 Frees (1970), 370.) Hybrid cells obtained after fusion of mouse myeloid 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 testing, clone VÚ 303/4 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 gG glycoprotein of the Herpes simplex virus type 2. The hybridoma VÚ 303/4 can be cultivated in vitro in media suitable for animal cells, or in vivo in the peritoneal cavity of a BALB/c mouse. Antibody production can be started from preserved frozen cells stored in liquid nitrogen without further immunization of the animal with the antigen.

Example

In order to obtain a larger amount of monoclonal antibody VU 303/4 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 (0.5 ml intraperitoneally per 1 mouse) 15 days before the application of cells. After 10 days of hybridoma growth in the peritoneal cavity, the mice were killed and the produced ascitic fluid was collected. This procedure can be used to obtain an average of about 7 ml of ascitic fluid containing 8 mg/ml of antibody. The ascitic fluid containing the hybridoma product VU 303/4 showed specific binding to Herpes simplex virus type 2 in radioimmunoassay (RIA) and in immunoenzymatic assay.

CS 270 193 Bl analysis (ELISA). The binding of a monoclonal antibody to the gG glycoprotein of the Herpes simplex virus type 2 was determined by the method of radioimmunoprecipitation and electrophoresis in polyacrylamide gel.

Hybridoma cells VÚ 303/4 grow in vitro as semi-suspension cultures. They have a round shape and size characteristic of myeloma cells. They contain fused cell nuclei and are aneuploid. Hybridoma cells VÚ 303/4 have an ultrastructural picture 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 ^(1959, 396). This medium, referred to as DMEM, is supplemented with gentamicin and inactivated precolostral calf serum (10%, Bioveta, Ivanovice na Hané) before culturing the hybridoma. 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 2a subclass. .

Hybridoma VÚ 303/4 can be used as a source of antibody to the gG glycoprotein of the Herpes simplex virus type 2, which can be used for qualitative detection of the presence of the Herpes simplex virus type 2 in the investigated material, for quantitative determination of the amount of infective virus or glycoprotein gG, in evaluating the epidemiological situation, for purification of glycoprotein gG from extracts of infected cells using immunoaffinity chromatography and as a source of antibody of a single subclass (IgG 2a) for the preparation of antisera specific to the above subclass.

Claims (1)

2 EN 270 193 B1 analysis (ELISA). By radioimmunoprecipitation and polyacrylamide gel electrophoresis, the binding of the monoclonal antibody to the Herpes simplex virus gG virus glycoprotein gi. 2 was enhanced in vitro in vitro as a semi-suspension culture. They have a round shape characteristic of myeloma cells. They contain fused cell nuclei, ane-uploid. V1303/4 hybridoma cells have an ultra-structural image of typical myeloma cells where free and membrane-bound polyribosomes are the predominant organelle. The basic culture medium is Dulbecco's modification of Eagle's minimal essential medium (Dulbecco, R., Freeman, G., Virology J3 (1959, 396). This medium, referred to as DMEM, is supplemented with gentamycin and inactivated precolostral gelling agent (10%, Bioveta) for hybridoma culture. The hybridoma is cultured at 37 ° C in an atmosphere of 5% CO 2 · Oe's generation time is approximately 24 h. The antibody produced is a monoclonal immunoglobulin of the IgG 2 subclass. gGvirus Herpes simplex type 2 glycoprotein, which can be used to quantify the presence of the Herpes simplex virus type 2 in the treated material, to quantitate the amount of infecting virus or glycoprotein gG, in assessing the epidemiological situation, for the purification of gG glycoprotein from infected cell extracts by immunoaffinity chromatography and as an antibody source area single subclass (IgG 2a) for preparing ántisérSpecifických for said subclass. OBJECT OF THE INVENTION MyS lymphocyte hybridoma VU 303/4, producing monoclonal antibody poritriody IgG 2a to glycoprotein gG Herpes simplex virus type 2.