EIA was done as described previously for other viral antibody determinations [18 (link)]. For coronavirus-specific antibody EIA, 96-well microtiter plates (Nunc Maxisorp, Thermo Fisher Scientific) were coated with 50 µL of GST-N (2.0 µg/mL), RBD-mFc-8×His (4.2 µg/mL), and S1-mFc-6×His (3.0 µg/mL) in PBS at 4°C overnight. GST (0.7 µg/mL) and proMstn-mFc-6×His (4.2 µg/mL) proteins were used as negative control antigens. After coating, the plates were washed once with washing buffer (0.05% Tween-20 in PBS). Serum samples were inactivated at 56°C for 30 minutes, and 100 µL of 1:300 diluted serum specimens in sample buffer (5% swine serum [Biological Industries], 0.1% Tween-20 in PBS), were incubated for 2 hours at 37°C. Horseradish peroxidase-labeled anti-human IgG (1:8000 dilution; Dako), anti-human IgA (1:8000 dilution; Invitrogen), or anti-human IgM (1:4000 dilution; Dako) antibodies in sample buffer was added and incubated for 1 hour at 37°C. TMB One (3, 3’, 5, 5’-tetramethylbenzidine, Kementec Solutions) was used as a substrate and after 20 minutes incubation at room temperature, the reaction was stopped with 0.1 M H2SO4. The absorbance was measured at 450 nm (Victor Nivo, PerkinElmer). The absorbance for negative control antigens was subtracted from the respective sample absorbance and the results were expressed as EIA units using a pool of negative serum samples (given a unit value of 0) and a pool of highly positive serum samples (unit value of 100) as standards. The unit values for SARS-CoV-2 GST-N–based EIA were adopted to other HCoV GST-N–based EIAs because no confirmed negative and highly positive control samples were available for SARS, MERS, and low-pathogenic coronavirus N antibody assays.
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