CS243617B1 - Method ofherpes simplex hsv-2 type specific virus preparation - Google Patents

Abstract

A method of preparing an HSV-2 antigen useful to detect type-specific antibodies HSV-2. The described method of preparation of HSV-2 antigen on a garden snail lectin column agarose 4B bound and eluted with 0.0M solution of N-acetylgalactosamine sugar to prepare specifically reacting HSV-2 antigen.

Description

(54) A method for preparing a herpes simplex virus type-specific HSV-2 antigen

A method for preparing HSV-2 antigen useful for detecting type-specific HSV-2 antibodies. The described method of preparation of HSV-2 antigen on agarose 4B snail lectin lectin column and elution of 0.0 M sugar solution of N-acetylgalactosamine allows to prepare specifically reactive HSV-2 antigen.

The invention relates to the preparation of a type-specific herpes simplex virus type 2 (HSV-2) antigen for the detection of type-specific antibodies against HSV-2.

Herpes simplex type 1 (HSV-1) oral and type 2 (HSV-2) genital are genetically related and most of their antigens cross-react. Currently used methods for determining the presence of specific HSV-2 antibodies in human sera use either indirect methods, eg neutralization assay (Rawls et al., 1970, J. Immunol. 104, 599-606), where by calculating the ratio anti-HSV-1 and anti-HSV-2 antibody titers (II / I index) determine the presence of HSV-2 antibodies by screening for type-common antibodies with cell extract (Porghani et al., 1975, J. Clin. Microbiol. 2, 410-418) ), by blocking cross-reacting antigens with heterotypic antibodies (Vestergaard et al., 1979, Acta Path. Microbiol.

Scand. 85, 261-263).

Current direct methods utilize electrophoretically isolated (Dreesman et al., 1979, Intervirology 12, 115-119) or radioimmunoprecipitated (Eberle and Courtney 1981, Inf. Immun. 31, 1062-1070) antigen HSV-2.

The disadvantage of these methods is lengthy, laborious, low efficiency. Olofsson and co-workers (1981, J. Virol. 38, 564-570) bound HSV-1 glycoprotein C to lectin of the glandular snail covalently bound to agarose and eluted with N-acetylgalactosamine solution.

It is an object of the present invention to remove the type-common antigens of herpes simplex virus type 1 (HSV-1) and herpes simplex type 2 (HSV-2), thereby allowing detection of HSV-2 antibodies in individuals infected with HSV-1 virus.

The invention solves the problem by using affinity chromatography on the snail lectin lectin to prepare a type-specific HSV-2 antigen. A suspension of infected HSV-2 cells with virus is applied to a column of lectin bound to agarose 4B. After washing the column with 10 volumes of 1% Triton in TRIS-HCl buffer solution (0.15M NaCl, 0.02M TRIS-HCl pH = 7.5), the antigen is released by adding a 0.01 M solution of N-acetylgalactosamine in TRIS-HCl buffer supplemented with 1 % Triton X-100. The antigen thus isolated can be used directly without further purification in a variety of serological tests (radioimmunoassay-RIA, enzymatic immunoassay-ELISA, radioimmunoassay-RIPA).

Description of a particular embodiment.

Suspensions of HSV-2 virus infected cells at high multiplicity of infection are mixed with a mixture of 1% sodium deoxycholate and 1% Tween 80 after washing with physiological phosphate buffer, and left at 37 ° C for 1 hour. The insoluble components were removed by centrifugation (100,000xg) for 1 hour and the supernatant was applied to a column of lecithin bound to agarose 4B. (A ratio of 4x10 6 cells per 0.1 ml column volume is preferred).

After washing the column with 10 volumes of 1% Triton in TRIS-HCl buffer solution (0.15M NaCl, 0.02M TRIS-HCl pH = 7.5), the antigen is released by adding 0.01M N-acetylgalactosamine solution again in TRIS-HCl-enriched buffer 1% Triton X-100. To detect type-specific antibodies, it is preferable to use the HSV-2 antigen thus prepared without further processing in the RIA or ELISA method.

243617.

Claims (1)

SUBJECT OF THE INVENTION Method for preparing a herpes simplex HSV-2 type-specific antigen, characterized in that a suspension of infected HSV-2 cells with the virus is applied to a column of agarose-bound snail lecithin after washing the column with 10 volumes of physiologically acceptable buffer at pH = 7.5 , preferably in 0.15M NaCl, 0.02M tris-hydroxymethyl-aminomethane supplemented with a non-ionic detergent, preferably 1% octylphenol-polyethylene glycol ether, the antigen is released by adding a 0.01M solution of N-acetylgalactosamine in a physiologically acceptable buffer at pH = 7, 5 supplemented with a nonionic detergent.