ELISA with colorimetric detection
Polystyrene microplates (Maxisorp, NUNC, Roskilde, Denmark) were coated overnight at 4 °C with 0.1μg purified Spike per well, in coating buffer, and washed five times with PBS. Blocking solution (200 μL/well) was added, and plates were incubated for 1 h. After washing six times, duplicate serum samples diluted 1/100 (100 μL/well) were added and incubated for 1 h. Microplates were washed six times and incubated for 30 min at 37 oC with anti-human IgG-biotin (diluted 1/180,000). Plates were washed six times, and bound antibodies were detected with Streptavidin-HRP (diluted 1/2,000, 30 min at 37 °C). After washing (five times plus one final wash with PBS), the chromogenic substrate was added and plates were incubated for 15 min in the dark. The color reaction was stopped with 2 M H2SO4. The oxidized substrate was measured at 450 nm with an ELISA plate reader MultiskanFC (Thermo Scientific Labsystems, USA). The blank control was made by replacing serum samples with PBS-MT. Results were calculated as specific absorbance (A = the mean of each sample minus the mean of the blank control or Ac), and expressed as Standard Deviation scores: SDs = (As-Ac)/SDc, where Ac is the mean specific absorbance of healthy control sera and SDc its standard deviation. The cut-off value of the assay was set at SDs = 3.0.