ZIKV Antibody Detection by ELISA

ZIKV E, EDIII, or hFc-specific antibodies in immunized mouse sera were analyzed by ELISA as previously described [30 (link)]. Briefly, ELISA plates were coated with ZIKV EDIII protein, ZIKV full-length E protein with a His₆ tag (Aviva Systems Biology, San Diego, CA, USA), or a C-terminal hFc-fused control protein containing a receptor-binding domain (i.e., RBD-Fc) of Middle East respiratory syndrome coronavirus (MERS-CoV) spike protein [31 (link)] (1 μg/mL) overnight at 4 °C, and blocked with 2% fat-free milk in PBST (PBS containing tween-20) at 37 °C for 2 h. The plates were washed with PBST for 3 times, and sequentially incubated with serial dilutions of mouse sera and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:5000) or IgG-Fab (1:3000) (for anti-ZIKV-E or anti-hFc antibodies), IgG1 (1:5000), or IgG2a (1:2000) antibodies (Thermo Fisher Scientific) at 37 °C for 1 h. The reaction was visualized after addition of 3,3′,5,5′-tetramethylbenzidine substrate (Sigma, St. Louis, MO, USA) and stopped with 1N H₂SO₄. Absorbance at 450 nm was measured using an ELISA plate reader (Tecan, Morrisville, NC, USA).