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Detection of Japanese Encephalitis Virus Antigens by the Sandwich ELISA in Infected Cell Culture Fluid and Cell Homogenates

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Title: Detection of Japanese Encephalitis Virus Antigens by the Sandwich ELISA in Infected Cell Culture Fluid and Cell Homogenates
Authors: Bundo-Morita, Keiko
Issue Date: 30-Jun-1989
Publisher: 長崎大学熱帯医学研究所 / Institute of Tropical Medicine, Nagasaki University
Citation: 熱帯医学 Tropical medicine 31(2). p49-65, 1989
Abstract: Sandwich ELISA was applied to detect Japanese encephalitis (JE) virus antigens in culture fluid and homogenate of Aedes albopictus, clone C6/36, and BHK21 cells daily after infection with various concentrations of JE virus. In both cell systems, the ELISA antigen in the infected fluid became detectable when infective virus titer rose up to 10^7 PFU/ml, and the ELISA titer remained at similar levels after reaching its plateau. Growth curve experiment of JE virus in both cell systems at high input multiplicity of infection showed that the titers of virus infectivity, hemagglutinating activity (HA), and ELISA antigen rose up almost in parallel in the infected BHK21 cell culture fluid and homogenate as well as in the infected C6/36 cell culture fluid. In the infected C6/36 cell homogenate, however, the HA titer remained at undetectable level while infectivity and ELISA titers rose up. Density gradient sedimentation analysis on the infected BHK21 and C6/36 cell culture fluids and homogenates showed that larger amounts of the virus antigen were detected in the slowly sedimenting light fraction (density 1.12 g/cc in potassium tartrate), with less amounts in the rapidly sedimenting heavy fraction associated with virus infectivity (densty 1.16 g/cc). The detection of JE virus ELISA antigen was applied to the culture fluid of C6/36 cells inoculated with field-caugth Culex tritaeniorhynchus mosquito homogenates and maintained at 28℃. The JE-ELISA antigen became detectable between 2 to 5 days after the inoculation for all the 9 pools which eventually turned out to be positive with JE virus isolation by the established method, while all the 11 pools which turned out to be negative with JE viurus isolation did not produce detectable levels of JE-ELISA antigen except a single specimen showing transient and borderline level (1 unit) of the reaction. A total of 256 culture fluids from C6/36 cell cultures inoculated with field-caught Cx. tritaeniorhynchus between 1978 to 1981 were assayed for their JE-ELISA antigen titers. All the 110 fluids, which were positive with JE virus isolation, possessed detectable levels (more than 4 units except a single specimen with 2 units) of JE-ELISA antigen, in contrast to 146 fluids, which were negative with JE virus isolation, with less than 2 units of JE-ELISA antigen titers. By applying the sandwich ELISA, the procedure for screening JE virus isolation by Aedes albopictus clone C6/36 cells could be reduced to 5 days in contrast to the previous procedure requiring total 10 days of incubation.
URI: http://hdl.handle.net/10069/4543
ISSN: 03855643
Type: Departmental Bulletin Paper
Appears in Collections:Volume 31, No. 2

Citable URI : http://hdl.handle.net/10069/4543

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