ELISA Protocol for SARS-CoV-2 RBD Antibody Detection

In short, microwells of ELISA plates (Corning, NY, USA) were coated with 50 ng RBD protein each (Genscript, Nanjing, China) at 4 °C overnight. Plates were blocked with 10% FBS in PBS for 1 h at 37 °C, and then incubated with 2-fold serially diluted serum samples for another hour. After washing three times, plates were incubated with rabbit anti-mouse IgG-HRP antibody at a dilution of 1:20,000 (Abcam, Cambridge, UK) at 37 °C for 1 h. Tetramethylbenzidine (Solarbio, Beijing, China) and hydrogen peroxide were used for color development, and the reaction was stopped with 2 M H₂SO₄. The absorbance was measured at 450 nm, and an optical density at 450 nm (OD₄₅₀) value greater than 2.1-fold of the background value was regarded as positive [25 (link)].