Hcov hku1

The spike protein and S1 subunits of seasonal hCoVs (HCoV-OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E) were purchased from Sino Biological (catalog numbers 40607-V08B, 40607-V08H, 40606-V08B, 40602-V08H, 40604-V08B, 40600-V08H, 40605-V08B, and 40601-V08H, respectively). Furthermore, 100 µl of antigens at a concentration of 0.5 ug/ml was coated in 96-well ELISA plates overnight at 4°C. The plate was washed with PBS-T (0.05% Tween 20) five times and blocked with 250 ul of blocking buffer (PBS, ph 7.4, + 2% BSA + 5%milk) for 2 h at 37°C. The blocking solution was removed, and diluted serum samples from a starting concentration of 1:100 were incubated at 37°C for 1 h. The plates were washed five times with PBS-T and 100 μl HRP-goat anti-human IgG (1:5,000 dilution) in diluted blocking buffer for 1 h at 37°C. The plates were washed with PBST five times. Then, 100 ul of substrate was added in each well for 15 min at room temperature. The reaction was stopped by adding 50 µl of 1 nm H₂SO4 to each well, and the reading was taken at 450 nm.

Recombinant SARS-CoV-2 S1 subunit of the S protein (amino acids 1–685, expressed in mammalian HEK293 cells), MERS-CoV S1 subunit (amino acids 1–725, expressed in mammalian HEK293 cells), and full-length S proteins (expressed in baculovirus-insect cells) from hCoV-OC43, hCoV-NL63, hCoV-229E, and hCoV-HKU1 viruses tagged with histidine tag (His-tag) were purchased commercially (Sino Biological, China). Recombinant SARS-CoV-2 and MERS-CoV N proteins were expressed and purified from Escherichia coli BL21 (DE3) cells using a nickel-nitrilotriacetic acid (Ni-NTA) column according to the manufacturer's protocol and as previously described^{7 (link)}. Positive fractions of N proteins were pooled, aliquoted, and stored at − 80 °C until used. SARS-CoV-2 proteins were confirmed by Western blot using anti-His tag antibodies as well as SARS-CoV-2 seropositive and seronegative human serum samples as previously described^{7 (link)}.