SARS-CoV-2 RBD mutants were purified and coupled onto magnetic multiplex beads (Luminex Corporation) as previously described and mixed in a bead cocktail (35 (link)). Bead cocktails were dispensed into each well of a black, clear-bottom, 384-well plate (Greiner), and mAbs at a starting concentration of 80 nM serially diluted were next added to the bead cocktail and incubated on a plate shaker for 2 hours at room temperature. The binding affinities of mAbs to different SARS-CoV-2 RBD mutants were detected using anti–human IgG-PE (Southern Biotech), and the mean fluorescence intensity (MFI) of the PE reporter was acquired on a FLEXMAP 3D machine (Luminex Corporation). To measure the competition of mAb-RBD binding by ACE2, bead cocktail and serially diluted mAbs were first mixed as described above, followed by the addition of Avi-tagged biotinylated ACE2. The plate was incubated on a plate shaker for 2 hours at room temperature. ACE2 binding was detected with streptavidin, R-PE conjugate (S866, Thermo Fisher Scientific) for 1 hour, followed by the addition of the R-PE Biotin-XX conjugate (P811, Thermo Fisher Scientific) for another 1 hour. The binding of ACE2 was detected as the MFI of the PE reporter using FLEXMAP 3D machine (Luminex Corporation).