SARS-CoV-2 Antibody Detection Assay

Binding plates (Costar) were coated with 100 ng/well Spike protein or Nucleocapsid protein (SinoBiological) at 4 °C overnight. The plates were blocked with 4% bovine serum albumin (BSA) for 90 min. After blocking, 50 μL of serum dilution was added to each well and incubated at 37 °C for 1 h. The plates were washed with PBS containing 0.05% Tween-20. Enzyme-labeled goat anti-human IgG-Fc or enzyme-labeled goat anti-human IgM-Fc was added as the secondary antibody. After incubation for 45 min, the plates were washed, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added, and then 100 μL of 2 mol/L H₂SO₄ was added to stop the reaction. The microplates were read using a microplate reader (Multiskan FC, Thermo Scientific) at 450 nm. The mean HC area under the curve (AUC) value + 3SD was used as the cutoff value to determine positive serum samples [6 (link)].