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.