Fortgeschrittene Techniken in Biologie und Medizin
Offener Zugang
ISSN: 2379-1764
ISSN: 2379-1764
Mohammad M Hossain* and Raymond RR Rowland
Classical swine fever virus (CSFV) E2 glycoprotein was expressed in an alphavirus based replicon particles (RP) expression system. A fluorescent microsphere immunoassay (FMIA) has been developed for the detection of CSFV E2-specific antibody in swine vaccinated with alphavirus RP. CSFV full length E2 (aa 1-376) was fragmented into several pieces and recombinant proteins were expressed in Escherichia coli. Purified proteins were conjugated to microsphere beads, the target antigens were assembled into a single multiplex, and tested against sera vaccinated with alphavirus-expressed antigens. The results reported as mean fluorescence intensity (MFI) obtained from the median value for at least 100 microspheres and the MFI values converted to positive per sample (S/P) ratio. Of the eight recombinant E2 proteins evaluated in this study, the highest MFI values were obtained for E2 (aa 1-181). CSFV E2 glycoprotein was expressed in alphavirus based replicon expression system. The results show that vaccinated animals had CSFV-specific IgA, IgG and IgM in serum and oral fluids. The MFI values for the negative serum sample showed 20-70-fold reduction compared to positive serum sample. Antibody response to CSFV antigens were IgG>IgM>IgA. The results demonstrated that the simultaneous detection of IgG, IgM and IgA antibodies could provide an improved diagnostic tool.