Alexa fluor 647 conjugated to goat anti human or anti mouse igg

Vero cells were inoculated with ZIKV at an MOI of 1. After 24 h, the cells were washed in PBS, detached after incubation in 10 mM EDTA in PBS for 15 min at 37 °C, and then washed again in chilled PBS, 4 mM EDTA, 0.4% BSA (fluorescence-activated cell sorting (FACS) buffer). The cells then were incubated with serial dilutions (20 μg/mL to 2 pg/mL) of each anti-NS1 or an isotype control mAb for 1 h at 4 °C. After washing in FACS buffer, cells were stained with Fixable Viability Dye eFluor 506 (eBioscience) and Alexa Fluor 647 conjugated to goat anti-human or anti-mouse IgG (Thermo Fisher). Cells were washed before fixing in 4% PFA in PBS for 10 min and then processed on a MACSQuant Analyzer (Miltenyi Biotec) using FlowJo software. After gating on live cells, the percent reactivity to cell surface-expressed NS1 was determined and plotted for each dilution of mAb (Supplementary Fig. 2a). The EC₅₀ of binding value was calculated using a 4-parameter logistic curve.

Vero cells were inoculated with WNV at a multiplicity of infection (MOI) of 5. After 24 h, the cells were washed in PBS and detached by incubation in PBS containing 10 mM EDTA for 15 min at 37°C. The cells were washed in chilled FACS buffer and pelleted by centrifugation at 300 × g for 3 min at 4°C. Subsequently, the cells were resuspended in serial dilutions (20 μg/ml to 2 pg/ml) of MAb (anti-NS1 or isotype control) for 1 h at 4°C. After washing in FACS buffer, cells were stained with fixable viability dye eFluor 506 (1:1,000 dilution; eBioscience) and Alexa Fluor 647 conjugated to goat anti-human or anti-mouse IgG (1:2,000 dilution; ThermoFisher). Cells were washed again before fixing in 4% PFA in PBS for 10 min and then processed on a MACSQuant analyzer (Miltenyi Biotec). After gating on live cells, the percent reactivity to cell surface-expressed NS1 was determined for each dilution of MAb. The EC₅₀ of binding to cell surface NS1 was calculated using a 4-parameter logistic curve.