2.3. Detection of IgM and IgG anti-RBD and -NP antibodies by enzyme-linked immunosorbent assay (ELISA)
For ELISA assays, we used recombinant SARS-CoV-2 NP protein expressed in Baculovirus-insect cells (Cat# 40588-V08B, Sino Biological Inc, China), and RBD expressed in HEK293 cells (Cat# 40592-V08H, Sino Biological Inc). RBD (1.0 µg/ml) or NP protein (1.5 µg/ml) were coated in flat-bottom 96-well Maxisorp microtiter plates (Nunc, Denmark) in phosphate-buffered saline (PBS) (pH 7.4) overnight at 4°C. After blocking with 3% w/v skimmed milk (Sigma Aldrich) in PBST (0.05% v/v Tween‐20 in PBS, Sigma-Aldrich) as the blocking buffer, 1:200 diluted sera were applied onto the plates in the blocking buffer and incubated at 37°C for 1 h, followed by three washes with PBST. Subsequently, plates were incubated with horse-radish peroxidase (HRP)-conjugated mouse monoclonal anti-human IgM or IgG (produced in our lab) at 1:1000 and 1:2000 dilution, respectively in the blocking buffer at 37°C for 1 h. The reactions were developed by the addition of tetramethylbenzidine (TMB) substrate solution (Pishtaz Teb, Iran) for 15 minutes and stopped by the addition of 1M H2SO4. Then, the optical density (OD) of the reactions was measured at 450 nm wavelength minus 630 nm using a microplate reader (Biotek, USA). The quantitative cut-off value for seropositivity for COVID-19 was defined as the mean OD of healthy samples plus 2 SDs.