Biotinylated goat anti human or goat anti mouse igg h l

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).

Fifty nanograms of WNV, JEV, DENV2, ZIKV, YFV, or TBEV NS1 proteins (all from Native Antigen) were immobilized on MaxiSorp microtiter plates (Nunc) overnight at 4°C in 50 μl of sodium bicarbonate buffer, pH 9.3. The plates were washed four times with PBS and blocked with ELISA buffer by incubation for 1 h at 37°C. Subsequently, the plates were incubated with MAb (anti-WNV NS1 or isotype control) diluted in ELISA buffer for 1 h at room temperature. For cross-reactivity ELISAs, the plates were incubated with MAb at 1 μg/ml; for avidity studies, the plates were incubated with serial dilutions of MAb as indicated in the figures. After washing four times in PBS containing 0.05% Tween 20, the plates were incubated with biotinylated goat anti-human or goat anti-mouse IgG (H+L; 1:2,000 dilution; Jackson ImmunoResearch) for 1 h. The plates were washed again and incubated with streptavidin-conjugated horseradish peroxidase (1:625 dilution; Vector Laboratories) for 15 min. After a final wash series, the plates were developed, and absorbance was read as described above. For avidity studies, the EC₅₀ of binding to solid-phase NS1 was calculated using a 4-parameter logistic curve.