2.4. Peptide-based ELISA
Preliminary peptide-based ELISA was performed using pooled sets of three
peptides. ELISA was performed according to the protocol described in
section 2.3. with the following modifications. In brief, flat-bottom
96-well Maxisorp plates (Nunc) were coated with 3.5 µg/ml of each
peptide in 3 peptide pool sets, native proteins (1.0 µg/ml of RBD or 1.5
µg/ml NP protein) or 1% 2-Mercaptoethanol (2-ME, Sigma Aldrich) reduced
proteins in PBS and incubated overnight at 4 °C. After washing three
times with 0.05% PBST, the plates were blocked using blocking buffer
for 1 h at 37 °C, before the addition of serum samples at 1:200 dilution
in the blocking buffer for 1 h at 37°C. HRP-conjugated mouse monoclonal
anti-human IgG antibody was used at 1:2000 dilution for detection of
peptide‐bound antibodies. Development was performed by the addition of
TMB and stopped with 1M H2SO4.