CS271293B1 - Mouse lymphocyte hybridoma vu t51/3 - Google Patents

Abstract

The solution applies to the murine lymphocytic hybridoma that produces the monoclonal antibody against the virus Herpea simplex type 1 and is stored in the collection of hybridomas of the Institute of virology SAV in Bratislava under the title V' T51/3.The monoclonal antibody produced by the hybridoma V' T51/3 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.e. a hybrid one-cell organism constructed by fusing a mouse myeloma cell Sp2 / O and a mouse spleen lymphoid cell producing an antibody to Herpes simplex virus type 1.

In Herpes simplex virus, we recognize two antigenically different types of Herpes simplex type 1 and type 2. , Schmidt N., Lennette E. H: Solid phase radioimmunoassay for identification of herpesvirus hominis types 1 and 2 from clinical material Appl. Microbiol · 28, / 1974/661 - 667 * Dreesman GR, Watson 0. 0, Courtney R. Adam, Adam E Mel, Melnick L L .: Oetection of herpesvirus type- ⁴

The serum of such immunized animals, taken after multiple doses of antigen, served as a source of so-called type-specific antibodies which were used to prove the Herpes simplex virus type antigen. · Since Herpes simplex viruses type 1 and 2 contain not only type-specific, but also type-common antigenic determinants, the herpes simplex virus antisera thus prepared contain high levels of type-common antibodies that reduce or prevent completely Correctly identifying the type of infecting virus in diagnostic testing · Type-common antibodies are usually removed from Herpes simplex type 2 virus sera, which is a very material-intensive procedure and, in a small number of cases, successful · Production batches of conventional typespecific h Antisera are easy to standardize and live in a wide quality range · Recently, monoclonal antibodies, so-called type-specific monoclonal antibodies that are capable of detecting Herpes simplex type 1 virus infection, have been successfully used to clone the virus.

The above disadvantages of the methods used so far do not occur if there is also a hybridoma bank line producing a type-specific monoclonal antibody against Herpes simplex virus type 1, which is stored in the collection of hybrids of the Institute of Virology of the Slovak Academy of Sciences, Mlýnská dolina 1, Bratislava.

The hybrids were obtained in a manner 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 · Monoclonal antibodies: A new dimension in Biological analyzes · Kennett R.H. et al., eds New York, Diapers Press (1980), 37C) · Hybrid cells obtained after fusion of murine myeloma Sp2 (0 cells and cells obtained from the spleen of BALB (c mice immunized) with an extract of cells infected with Herpes simplex virus type 1, were also cloned, and after testing a clone of TU T51 / 3 was selected. a monoclonal antibody capable of specifically reacting with Herpes simplex virus type 8 virus. T51 / 3 hybrid hybrids can be cultured in vitro in animal cell media or in vivo in the perritoneal cavity of a BALB / c mouse. From canned frozen cells stored in liquid nitrogen , the production of an antibody without further immunization with an antigen may have begun.

In order to obtain a greater amount of monoclonal antibody ν51 T51 / 3 by culturing hybridomas of cells in vivo, 5x10 ⁶ cells were injected into the peritoneal cavity of mice. For better cell attachment, mice were pre-treated with paraffin oil (0.5 ml anthraperitoneally per mouse) 15 days prior to cell application, after 10 days of hybridoma growth in the peritoneal cavity, the mouse was killed and the ascites fluid produced was collected. On average, about 7 ml of ascites fluid containing 8 mg / ml antibody is obtained by this procedure. The ascites fluid containing the hybridoma product T51 / 3 showed specific binding to Herpes simplex virus type 1 in the radioimmunoassay (RIA), immunoenzymatic assay (ELISA) and immunofluorescence assay (IF).

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In vitro hybridoma cells of T51 / 3 growth as a semi-suspension culture. They have a round shape and size characteristic of myeloma cells. They contain fused cell nuclei, are aneuploid. The basic culture medium is Dulbecco's modification of Eagle's minimal essential medium (Dulbecco, R., Freeman, G., Virology 8 (1959), 396). This medium, called OMEM is the cultivation of the hybridoma supplemented with gentamicin and inactivated precolostral calf serum (10%, Bioveta Ivanovice na Hane) · The hybridoma was cultured at 37 ° C in 5% CO ^ · 3 ^eho of recent time is about 24 h · the antibody produced is immunoglobin monoclonals of subclass igG 2b ·

The T51 / 3 hybridoma can be used as a source of antibody to Herpes simplex virus type 1, which can be used to qualitatively prove the presence of Herpes simplex virus type 1 in the material under investigation, quantitatively determining the amount of infecting virus, assessing the epidemiological situation and a single subclass (igG 2b) for preparing antisera specific for said subclass.

Claims (2)

Murine lymphocyte hybridoma TU / T51 / 3, producing a monoclonal antibody of subclass igG 2b against Herpes simplex virus type 1.