ZIKV EDIII-specific Antibody Analysis

Mouse sera were analyzed for ZIKV EDIII-specific antibody responses by ELISA as previously described^{32 (link),35 (link)}. Briefly, ELISA plates were coated with ZIKV EDIII-His or E1-454 protein overnight at 4 °C and blocked with 2% fat-free milk for 2 h at 37 °C. Serially diluted mouse sera were added to the plates and then incubated for 1 h at 37 °C. After washing, the plates were incubated with HRP-conjugated anti-mouse IgG (1:5000), anti-mouse IgG1 (1:2000, Bethyl Laboratories, Montgomery, TX, USA), or anti-mouse IgG2a (1:2000, Invitrogen) for 1 h at 37 °C. The reaction was detected by 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (Invitrogen) and stopped by 1 N H₂SO₄. The absorbance at 450 nm was measured using an ELISA plate reader (Tecan, Morrisville, NC, USA), and the respective antibody titers were calculated.