Spike ELISA
The ELISA protocol was adapted from Stadlbauer et al. (2020) using spike protein with >95% purity. Microtiter plates (96-well) were coated overnight with 50 μL of spike per well (2 μg/mL in PBS pH 7.4) at 4°C. The coating solution was removed and 300 μL of blocking solution (3% non-fat milk in 0.1% PBS-T) was added for 1 h and washed 3 times (with 0.1% PBS-T). Samples were added at 1/20 dilution and incubated for 2 h at RT. Plate was washed 3 times and 100 μL of anti-human IgG conjugate was added to the wells and incubated for 1 h at RT. Plate was washed 3 times and 100 μL of substrate was added and incubated in the dark for 45 minutes. The reaction was stopped by the addition of 50 of µl 3 M HCl and the plate was read at 490 nm using the Agility (Dynex Technologies) ELISA system. The index value was calculated as follows:
\(Index\ value=\frac{\text{Sample~{}OD}}{Mean\ of\ negative\ controls+3SDs}\)(Eq. 1)
The cut-off value was calculated with 100% specificity using ROC curves of calculated index values.