3 3 5 5 tetramethylbenzidine substrate

Recombinant NS1 proteins (0.4 μg/mL) were immobilized onto MaxiSorp 96-well plates (Thermo Fisher) overnight in 50 μL of sodium bicarbonate buffer, pH 9.3. For mAb domain mapping, full-length ZIKV NS1 (residues 1–352) or ZIKV NS1 DII/III (residues 172–352)^{35 (link)} was immobilized. For determination of cross-reactivity, ZIKV, DENV2, WNV, JEV, TBEV, or YFV NS1 proteins (all from Native Antigen) were adsorbed to wells of MaxiSorp microtiter plates overnight at 4 °C. Subsequently, plates were washed four times with PBS and blocked with ELISA buffer (PBS, 1% BSA, and 0.05% Tween 20) for 1 h at 37 °C. Plates then were incubated with anti-NS1 or isotype control mAbs diluted in ELISA buffer for 1 h at room temperature. After washing four times with ELISA buffer, plates were incubated with biotinylated goat anti-human or goat anti-mouse IgG (H + L; 1:2000 dilution; Jackson ImmunoResearch) for 30 min. Plates were washed again and incubated with streptavidin-conjugated horseradish peroxidase (1:625 dilution; Vector Laboratories) for 30 min. After a final wash series, plates were developed using 3,3′,5,5′-tetramethylbenzidine substrate (Agilent). The reaction was stopped using 2 N H₂SO₄ and absorbance at 450 nm was read with a TriStar Microplate Reader (Berthold Technologies).

MaxiSorp 96-well microtiter plates (Nunc) were coated overnight at 4°C with 20 ng of recombinant WNV NS1 protein (Native Antigen) in 50 μl of sodium bicarbonate buffer, pH 9.3. Subsequently, plates were washed four times with PBS and blocked with ELISA buffer (PBS containing 2% BSA and 0.05% Tween 20) for 1 h at 37°C. Plates then were incubated with anti-WNV NS1 murine MAbs at 10 μg/ml for 1 h at room temperature. Without washing, anti-WNV NS1 human MAbs were added to the plates at preoptimized concentrations and incubated for 10 min at room temperature. The plates then were washed four times in PBS containing 0.05% Tween 20 and incubated with goat anti-human IgG conjugated to horseradish peroxidase (1:2,000 dilution; Jackson ImmunoResearch 109-035-088) for 30 min at room temperature. After washing, plates were developed using 3,3',5,5'-tetramethylbenzidine substrate (Agilent) for 5 to 10 min. The reaction was stopped using 2 N H₂SO₄, and absorbance (450 nm) was read using a TriStar microplate reader (Berthold Technologies).