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.