ELISA
ELISA was performed to detect the binding of SARS-CoV-2 RBD protein to sACE2 receptor, as previously described.27,32,34 SARS-CoV and MERS-CoV RBDs, as well as sDPP4 protein, were used as controls. Briefly, ELISA plates were precoated with SARS-CoV-2, SARS-CoV, or MERS-CoV RBD (1 μg/ml) overnight at 4 °C and blocked with 2% fat-free milk in PBST for 2 h at 37 °C. Serially diluted sACE2, or sDPP4, protein was added to the plates and incubated for 2 h at 37 °C. After four washes, the bound protein was detected using hACE2- or hDPP4-specific goat antibody (0.5 μg/ml, R&D system) for 2 h at 37 °C, followed by incubation with HRP-conjugated anti-goat IgG antibody (1:5,000, Thermo Fisher Scientific) for 1 h at 37 °C. The reaction was visualized by addition of substrate 3,3’,5,5’-Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO) and stopped by H2SO4 (1 N). The absorbance at 450 nm (A450) was measured by an ELISA plate reader (Tecan, San Jose, CA).
The cross-reactivity of SARS-CoV-2 RBD protein to SARS-CoV RBD-specific antibody was performed by coating ELISA plates with SARS-CoV-2 RBD (1 μg/ml), as well as SARS-CoV RBD or MERS-CoV RBD (as controls, 1 μg/ml), followed by sequential incubation with serially diluted SARS-CoV RBD- or MERS-CoV RBD-immunized mouse sera and HRP-conjugated anti-mouse IgG (1:5,000; Thermo Fisher Scientific) antibodies.