Zika Virus Envelope Protein IgG ELISA Assay

Recombivirus^TM Mouse anti-ZIKV envelope protein IgG ELISA kits (Alpha Diagnostic International, RV-403120-1) were used according to the manufacturer’s protocol. In brief, 96-well plates coated with the ZIKV envelope protein were equilibrated with 300 μL of kit working wash buffer. Serial dilutions (3-fold) of sera from vaccinated mice were added. Diluted sera were incubated at RT for 1 h, and after four times washing buffer incubations, 100 μL/well of anti-mouse IgG HRP-conjugate working solution was added for 30 min at room temperature. Plates were washed 5 times and developed for 15 min at room temperature with 100 μL of (TMB) substrate (3,3′,5,5′-tetramethylbenzidine), then stopped by the addition of 100 μL of stop solution. Absorbance was measured at 450 nm on a microplate reader. ELISA ODs were compared between all vaccinated groups at different sera dilutions. For ELISA in BALB/c mice before challenge, antibody endpoint titres are the highest reciprocal serum dilution that resulted in an absorbance >2-fold over the background values, as calculate elsewhere [36 (link)]. Anti-Zika envelope antibody concentrations in A129 mice sera before challenge was measured by IgG ELISA using Zika Env antigen as previously described [48 (link)].