Detection and Quantification of Japanese Encephalitis Virus Antigen by ELISA

• Main Authors: LAN, QIAO-RU, 藍巧如
• Other Authors: KUO, TSUN-YUNG
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/suxcm4

碩士 === 國立宜蘭大學 === 生物技術與動物科學系生物技術碩士班 === 107 === Japanese encephalitis virus (JEV) is one of the important pathogenic viruses causing infection of the central nervous system. Although symptoms of Japanese encephalitis are rare, the case-fatality rate among those with encephalitis is high. Furthermore, survivors suffer severe permanent neurologic or psychiatric sequelae. Currently, two types of vaccines have been used in Taiwan. One is live-attenuated chimeric Japanese encephalitis vaccine and the other is Vero cell culture-derived inactivated Japanese encephalitis vaccine. In the vaccine manufacturing process, rapid test of the amount of antigen in bulk and finished product is a key parameter for vaccine formulation. Therefore, we established a Q-ELISA platform that could detect and quantify JEV antigen. JEV was cultured in serum-free medium, then purified and inactivated in order to prepare antigen to immunize rabbit for polyclonal antibodies (pAbs). The pAbs were verified to recognize JEV by immunofluorescence assay (IFA). Simultaneously, mice were immunized with purified antigen for hybridoma preparation from splenocytes. Three clones of hybridoma were selected after screening, and their binding affinity was confirmed by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Isotype analysis by mouse monoclonal antibody isotyping test kit showed that the hybridoma isotypes were IgG1 with kappa light chain. In addition, the anti-JEV mAb and rabbit anti-JEV pAbs were used for the development of quantification ELISA (Q-ELISA) to quantify JEV antigen. In this study, the best range of antigen detection was from 2500 ng/ mL to 156 ng/ mL (R2 values 0.99) when using anti-JEV mAb as capture, rabbit anti-JEV pAbs as detector and goat anti rabbit conjugated HRP as secondary antibody. We have shown that this platform can be successfully applied to detect JEV antigen from cultivated viral fluid.