A, The NS1 standard curve as determined by the purified rWNV-NS1 antigen-capture ELISA. Various concentrations of WNV-NS1 and DENV1-4-NS1 proteins were analysed. BSA was used to establish the baseline. B, The detection of NS1 in WNV-infected cell culture supernatants and other closely related members of the flavivirus family, DENV, JEV, YFV, and TBEV, by the NS1 antigen-capture ELISA. Two-fold serially diluted supernatants from WNV-, four DENV serotype-, JEV-, YFV-, or TBEV-infected cells were subjected to the WNV-NS1 antigen-capture ELISA. Data points represent the mean ± standard deviation from three replicates. The cut-off value was set at twice the average value of the negative controls from three replicates. Positive OD450 values were only observed for the WNV-NS1 protein and WNV-infected cell supernatants.