Enzyme-linked immunosorbent assays (ELISAs) were performed as described previously (8 (link)). Briefly, maxisorp plates (Nunc) were coated overnight at 4°C with 100 ng per well S or RBD protein in PBS. Plates were blocked with 100 μl of casein in PBS (Thermo Fisher Scientific) for 1 hour at room temperature. Serum serially diluted two times in casein in PBS was incubated at room temperature for 1 hour. Antibodies were detected using affinity-purified polyclonal antibody peroxidase-labeled goat–anti-monkey IgG (SeraCare, 074-11-021) in casein followed by 3,3′,5,5′-tetramethylbenzidine 2–component peroxidase substrate (SeraCare, 5120-0047). The reaction was stopped using stop solution (SeraCare, 5150-0021) and read at 450 nm. All wells were washed four times with PBS with 0.1% Tween 20 in between steps. Threshold for positivity was set at three times the optical density value of negative control (serum obtained from NHPs before start of the experiment) or 0.2, whichever was higher.
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