Development And Application Of Autologous Fusion Viral Capsid Antigens Of Epstein-Barr Virus

Objective To develop enzyme-linked immunosorbent assay(ELISA) kit for detection of IgG and IgM antibodies to viral capsid antigen(VCA) of Epstein-Barr virus(EBV) by using autologous viral capsid proteins.Methods Full-length p23(amino acids 1-162) and carboxy half of p18 (105-176) gene fragment were connected by a(Gly₄Ser)₃ linker using overlap extension PCR method.The fusion gene p23-p18 was inserted into the prokaryotic expression plasmid pThioHis A.The recombinant fusion protein was induced to express by IPTG in E.coli BL21 and purified by nickel-chelating Sepharose resin affinity chromatography.Its antigenicity and specificity was identified by Western blot.Fusion proteins with optimal dilution,which titrated against standard known anti-VCA positive and negative control sera,were used as the antigens for ELISA detection of IgG and IgM antibodies to VCA.Four hundred and fifity serum samples were examined for the presence of VCA-specific IgG and IgM by our kits,IFA and NOVATEC's VCA IgG and IgM ELISA Kit simultaneously.Comparing the results of our kits with that of IFA and NOVATEC's kits,the specificity,sensitivity, Youden's index,predictive value positive and predictive value negative of our kits were detected.The repeatability and stability of our kits were also detected.Results p23-p18 fusion gene was constructed correctly and expressed successfully in the prokaryotic expression plasmid.The fusion proein showed good reactivity with VCA IgM and IgG positive sera in Western blots.Compared with IFA, the sensitivity,specifity,Youden's index,coincident rate,predictive value positive and predictive value negative of the VCA IgG ELISA kit based on p23-p18 fusion antigens were 97.3%,97.4%,0.947,97.3%,99.8%and 77.6%respectively,and that of VCA IgM ELISA kit were 94.8%,99.5%,0.943,98.7%,97.3%and 98.9% respectively.Our ELISA kits had a good agreement with commercial kits and showed high repeatability and stability in the detection of VCA-sepecific IgG and IgM in clinical samples.Conclusion The ELISA based on fusion viral capsid proteins is sensitive, sepecific,simple and reliable test for the measurement of IgG and IgM antibodies to VCA of EBV for both clinical diagnosis and epidemiology studies of EBV infection.