SARS-CoV-2 Antibody Detection ELISA Assay

In this study, two in-house enzyme-linked immunosorbent assay (ELISA) systems were used to assess the participants’ anti-spike IgG (anti-S1 + RBD IgG) and anti-N IgG antibody levels [17 (link),18 (link),19 (link)]. Briefly, ELISA plates were pre-coated with SARS-CoV-2 Spike 1 and RBD proteins (Sino biological, Beijing, China) at a ratio of 6:4, and SARS-CoV-2 nucleocapsid protein (Sino biological, Beijing, China) for anti-spike IgG (anti-S1 + RBD IgG) and anti-N IgG detection. After the serum separation, the serum was diluted (1:100) with dilution buffer and dispensed into wells with positive, negative, and plate controls. After incubation for 15 min and a washing step, horseradish peroxidase-conjugated anti-human IgG (The NativeAntigens, London, UK) at a 1:4000 dilution was added to the wells. After a short incubation and wash, the substrate, 3,3′,5,5′-Tetramethylbenzidine (TMB), was added to each well, followed by a stop solution. The optical density of the final reaction was measured at 450 nm. The antibody level was finally determined by analyzing the OD/cut-off ratio.