Competitive ELISA for Epitope Mapping

For competitive ELISA used in epitope mapping of mAbs, 2 μg/mL recombinant RBD-his (Sino Biological, Beijing, China) was added in 384-well plates and incubated at 4 °C overnight. 50 μg/mL mAbs per well were added. The plates were incubated at 37 °C for 1 h and then washed. Biotinylation of mAbs (the top 20 neutralizing Abs and 81A11, previously reported SARS-CoV CR3022^{24 (link)}) were performed using the EZ-link NHS-PEO Solid Phase Biotinylation Kit (Pierce) according to the manufacturer’s protocol and purified using MINI Dialysis Unit (ThermoFisher, 69576). 500 ng/mL biotinylated mAbs were added to each well, and the plates were incubated at 37 °C for 1 h. ALP-conjugated streptavidin (Mabtech, Sweden, 3310-10) was added at 1:1000, followed by an incubation of 30 mins at 37 °C. For the quantification of bound IgG, PNPP (Thermo Fisher) was added at 1 mg/mL and the absorbance at 405 nm was measured by the MultiSkan GO fluoro-microplate reader (Thermo Fisher).