SARS-CoV-2 Seroprevalence Determination

To determine the seroprevalence, we utilized an in-house ELISA test that we recently developed and validated [9 (link)] to measure SARS-CoV-2 IgM and IgG antibodies against the most immunogenic SARS-CoV-2 antigens, nucleocapsid protein (N) and spike glycoprotein (S). Briefly, commercial recombinant SARS-CoV-2 S1 subunit (amino acids 1–685) (Sino Biological, Beijing, China), and an in-house produced recombinant SARS-CoV-2 N protein were used to coat 96-well ELISA plates at 1 μg/mL and 4 μg/mL in phosphate-buffered saline (PBS), respectively, for overnight at 4 °C. Plates were then washed with PBS with 0.05% tween-20 (PBS-T). After blocking the plates with 5% skim milk in PBS-T buffer at 37 °C for 1 h, 1:100 diluted serum samples were incubated for 1 h at 37 °C and washed. Anti-IgG and anti-IgM HRP-conjugated antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were then added for 1 h at 37 °C. Following incubation, plates were washed and 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (KPL, Gaithersburg, MD, USA) was added for 30 min in the dark at room temperature, and the reaction was terminated by 0.16 M sulfuric acid. Absorbance was measured at 450 nm using the ELx808 microplate reader (BioTek, Winooski, VT, USA). Cutoff values for the ELISA were 0.4 for IgG N-ELISA, 0.55 for IgM N-ELISA, 0.17 for IgG S1-ELISA, and 0.3 for IgM S1-ELISA as previously determined [9 (link)].