To determine the binding affinity of antibody to SARS-CoV-2 virus, virus was captured onto plates coated with mouse anti-SARS-CoV-2 spike (mAb31 with murine Fc) and then incubated with serial dilutions of SARS-CoV-2-specific human mAbs (full length IgG or Fab) followed by ALP-conjugated anti-human IgG (A8542, Sigma). The reaction was developed with PNPP substrate and stopped with NaOH. The absorbance was measured at 405 nm.

Results are expressed as the percentage of total binding, with 100% binding determined from the Ab concentration that gave maximum absorbance. GraphPad PRISM software was used to perform nonlinear regression curve-fitting analyses of binding data to estimate dissociation constants (Kd). Percent occupancy at IC50 was determined using the following formula: Percent occupancy = BMax [Ab]/(Kd+[Ab]), where the BMax is percent maximal binding, [Ab] is the concentration of Ab required to reach 50% FRNT and Kd is the concentration of Ab required to reach half-maximal binding.

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