Goat anti human igg conjugated to hrp

ELISA plates were coated overnight at 4°C with 0.1 μg/mL of mouse anti-human IgG (human CH2 domain with no cross-reactivity to rhesus macaque IgG; clone R10Z8E9; BioRad) and then blocked for 2 hrs. The serum samples were assayed at 4-fold dilutions starting at a 1:100 dilution in ELISA diluent (PBS containing 1% heat-inactivated FBS and 0.2% Tween-20). Samples were incubated for 1 hr at ambient temperature and then removed, and plates were washed. Wells then were incubated for 1 hr with goat anti-human IgG conjugated to HRP (ThermoFisher Scientific) at a 1:20,000 dilution. Wells were washed and then incubated with TMB substrate (KPL) (100 μL/well) and incubated for 10 min followed by 1N hydrochloric acid stop the reaction (100 μL/well). Microplates are read at 450 nm with 650 nm subtraction with an OD₄₅₀ nm cut-off of 0.052 (Biotek Cytation reader). Human mAbs were quantified using Prism software, version 7.04 (GraphPad), to analyze sigmoidal dose-response (variable slope), using 1:1 mixture of rEBOV-520 LALA and rEBOV-548 IgG1 cocktail as a standard.