We performed ELISAs as previously described [15 (link)]. Briefly, spike proteins were suspended at 1 μg/ml in 1 × PBS. One hundred microliters of protein suspension was added to each well of a 96-well Nunc MaxiSorp ELISA plate and allowed to coat overnight at 4 °C for 16 h. Wells were washed three times with 300 μl of 1× PBS + 0.05% Tween20 (wash buffer) followed by blocking for 2 h at room temperature with 200 μl of 1× PBS + 0.05% Tween20 + 5% Non-fat dry milk (blocking buffer). Wells were washed again three times with 300 μl of wash buffer prior to addition of 100 μl of sample diluted in blocking buffer (serum samples were heat inactivated for 45 min at 56 °C and diluted at 1:400 in blocking buffer). Samples were incubated for 1 h at room temperature, then washed three times with 300 μl of wash buffer. One hundred microliters of 1-Step Ultra TMB Substrate (ThermoFisher) was added and the plate was incubated for 10 min prior to stopping the reaction with 1 N sulfuric acid (Stop Solution, ThermoFisher). Absorbance was read at 450 nm and 650 nm on a BioTek Epoch2 plate reader. The process is semi-automated through the use of a BioTek EL406 plate washer/dispenser and two BioStack 4 plate stackers to minimize plate-to-plate variation and increase throughput (see Klumpp-Thomas C, Kalish H et al. 2020 for detailed automation methods) [15 (link)].