384-well Maxisorp plates (Thermo Fisher) were coated overnight at room temperature with 3 μg/mL in 20mM Tris pH 8 and 150mM NaCl of SARS-CoV-2 S2P (Pallenson et al 2017) or SARS-CoV-2 A222V-D614G S2P, produced as previously described in Walls et al. (2020) (link). Briefly, Expi293F cells were transiently transcribed with a plasmid containing the spike protein and supernatant was clarified six days later prior to Ni Sepharose resin purification and flash freezing. Plates were slapped dry and blocked with Blocker Casein in TBS (Thermo Fisher) for one hour at 37°C. Plates were slapped dry and S2E12 (Tortorici et al., 2020 ) or S309 (Pinto et al., 2020 (link)) antibodies were serially diluted 1:3 with a starting concentration of 1000nM in TBST or NIBSC human plasma (20/130 https://www.nibsc.org/documents/ifu/20-130.pdf ) was serially diluted 1:3 starting at 1:4 of original concentration in TBST and added to the plate for one hour at 37°C. Plates were washed 4x with TBST using a 405 TS Microplate Washer (BioTek) followed by addition of 1:5,000 goat anti-human Fc IgGHRP (Thermo Fisher) for one hour at 37°C. Plates were washed 4x and TMB Microwell Peroxidase (Seracare) was added. The reaction was quenched after 1–2 minutes with 1 N HCl and the A450 of each well was read using a Varioskan Lux plate reader (Thermo Fisher).
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