CS271292B1 - Mouse lymphocyte hybridoma vu 440/2 - Google Patents

Abstract

The solution applies to the murine lymphocytic hybridoma that produces the monoclonal antibody against virus Herpea simplex type 2 and is stored in the collection of hybridomas of the Institute of virology SAV in Bratislava under the title V' 440/2.The monoclonal antibody produced by the hybridoma V' 440/2 is suitable for diagnostic purposes in the immunofluorescent test (IF), in the radio-immunoassay test (RIA) and in the immunoenzymatic analysis (ELISA) for the determination of the presence and the amount of viral antigen in the tested material.

Description

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

In Herpes simplex virus, we recognize two antigenically different types of Herpes simplex type i and type 2. Heretofore antibodies to the Herpes simplex virus type 2 have been prepared by immunizing experimental animals, most commonly rabbits, with purified or unpurified virus respectively. by some isolated protein (Forghani B., Schmidt N., Lennette EH: Solid phase radioimmunoassay for identification of herpesvirus hominis types and 2 from clinical material. Appl. Microbiol. 28, (1974) 661-667 * Dreesman GR, Watson D. 0, Courtney R. Adam., Adam E., Melnick □.:: Detection of herpesvirus type-specific antibody by microsolid-phase radioimmunometric assay; Intervirology 12, (1979) 115-119). The serum of the animals immunized in this way, taken after multiple doses of antigen, served as a source of antibodies. Type-specific types that have been used to detect the Herpes simplex virus type 2 antigen in basic research and immunodiagnostic practice. Since Herpes simplex viruses type 1 and 2 contain not only type-specific, but also type-common antigenic determinants, the herpes simplex virus antisera prepared in this way contain high levels of type-common antibodies that download or reject antibodies. completely make it impossible to correctly determine the type of infecting virus in diagnostic testing. Type-common antibodies are usually removed from sera by plating with Herpes simplex virus type 1, which is a very material-intensive procedure and, moreover, in only a few cases succeeds. The production batches of conventional types of specific antisera are difficult to standardize and have a wide quality range. Recently, monoclonal antibodies, so-called monoclonal antibodies, have been successfully used for typing Herpes simplex type 2 virus. Type-specific, capable of detecting Herpes simplex virus type 2 infection in clinical material.

The above disadvantages of the processes used hitherto do not occur if available! a hybridoma cell line producing a type-specific monoclonal antibody to 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 VU 440/2.

The hybridoma was obtained by a method known from the literature (Kohler, G., Milstein, C .: Continuous Cultures of Fused Cell Secreting Antibody of Predefined Specificity. Nature, 256, (1975), 495, Gerhard W., Fusion of Cells in Monoclonal Antibodies: A New Dimension in Biological Analyzes Kennett R. H et al., eds. New York, Diaper Press (1980), 370. Hybrid cells obtained following fusion of mouse myeloma Sp2 / 0. cells and cells obtained from the spleen of BALB / c mice immunized with Herpes simplex type 2 virus-infected extract of cells were cloned and cloned VU 440/2 after testing.

The advantage of the hybridone is that it produces a homogeneous antibody, the so-called. a monoclonal antibody capable of specifically reacting with Herpes simplex virus type 2. The VU 440/2 hybridoma can be cultured in vitro in animal cell media 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 antigen immunization.

example:

To obtain more of the monoclonal antibody VU 440/2 by culturing hybridoma cells in vivo, 5x10 6 cells were injected into the peritoneal cavity of the mouse.

For better cell attachment, mice were pre-treated with paraffin oil (0.5 ml intraperitoneally per mouse) 15 days prior to cell administration. After 10 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the ascitic fluid produced was collected. On average, about 7 ml of ascites fluid containing 8 mg / ml of antibody can be obtained by this procedure. The ascites fluid containing the VU 440/2 hybridoma product showed specific binding to Herpes simplex virus type 2 in the radioimmunoassay (RIA), immunoenzymatic assay (ELISA), and immunofluorescence assay (IF).

VU 440/2 hybridoma cells grown in vitro as a semi-suspension culture, having a round shape and size characteristic of myeloma cells. The fused cell nuclei are aneuploid. The VU 440/2 hybridoma cells have an ultrastructural pattern of typical myeloma cells where the predominant organelle is free and membrane-bound polyribosomes. The basic culture medium is Dulbecco's modification of Eagle's minimum essential medium (Dulbecco, R., Freeman, G., Virology £ (1959), 396). %, Bioveta, Ivanovice na Hané), The hybridoma is cultured at 37 ° C in 5% CO 2 atmosphere. Its generation time is approximately 24 h. Antibody Produced Is an IgG 1 monoclonal immunoglobulin.

The VÚ 440/2 hybridoma can be used as a source of antibody to Herpes simplex virus type 2, which can be used to qualitatively detect the presence of Herpes simplex virus type 2 in the sample, to quantify the amount of infecting virus when evaluating the epidemiological situation and subclasses (IgG 1) for preparing antisera specific for said subclass.

Claims (1)

It is a lymphocyte hybridoma VU 440/2 producing a monoclonal antibody of subclass IgG 1 to Herpes simplex virus type 2.