96 well high affinity elisa plates

To quantify ZIKV-reactive IgG, 96-well high-affinity ELISA plates (Costar) were coated with ZIKV E protein (1 mg/ml ZIKVSU-ENV, The Native Antigen) in 100 μl coating buffer (0.1 M NaHCO₃) overnight at 4°C and then blocked for 1 h at room temperature (RT) with 5% Blocker Casein in PBS (Thermo Fisher Scientific). Mouse serum samples were serially diluted three-fold from 1:30 to 1:65,610 in 1% bovine serum albumin (BSA)/PBS and added to the coated wells. As a positive control, 10 μg of the pan-flavivirus envelope protein-specific mAb 4G2 (Absolute Antibody) was diluted in 1% BSA/PBS and titrated three-fold in the same manner as for the sera. After 1.5 h incubation at RT, the wells were washed with washing buffer (0.05% Tween 20 in PBS) and then incubated with HRP-conjugated goat anti-mouse IgG (1:5000 in 1% BSA/PBS) for 1.5 h at RT. TMB chromogen solution (Thermo Fisher Scientific) was added to the wells, the reaction was stopped by addition of 2N sulfuric acid, and the absorbance at 450 nm was read on a Spectramax M2E microplate reader (Molecular Devices). The ZIKV-specific IgG endpoint titers were calculated as the reciprocal of the highest serum dilution that gave a reading twice the cutoff absorbance of the negative control (1% BSA/PBS).