Figure S2.

Concentration-Dependent Binding of V_H-Fc ab8 and ACE2-Fc to Cell Surface-Associated SARS-CoV-2 S, Evaluation of Competition of ACE2 and V_H ab8 by ELISA and FACS, and Test of the Conformation Integrity of RBD Mutants by Using a Polyclonal Antibody and Monoclonal Antibody CR3022, Related to Figures 1, 2, and 3

(A) Cells were incubated with serially diluted antibodies or ACE2-Fc and subsequently with PE conjugated anti-human Fc antibody for flow cytometry analysis. Percentage of PE-A+ cells were defined by the above gate strategy in FlowJ, representing the percentage of V_H-Fc ab8 and ACE2-Fc bound 293T-S cells. (B) ACE2 blocking V_H ab8 for binding to RBD by ELISA. RBD was coated to plate and 10 nM of V_H ab8 in the presence of gradient concentration of ACE2 was added. Binding was detected by HRP conjugated anti FLAG tag antibody. (C) ACE2 blocking V_H ab8 for binding to cell surface associated S. S transiently transfected 293T was incubated with 1 μM V_H ab8 in the presence of various concentration of ACE2 (his tag). Binding of V_H ab8 was detected by the PE conjugated anti FLAG tag antibody. (D) and (E) Binding of a mouse polyclonal anti-SARS-CoV-2 RBD antibody and IgG1 CR3022 to the RBD mutants. RBD mutants were coated to plate and two concentrations of polyclonal anti-RBD antibody and CR3022 were added. Binding was detected by HRP conjugated anti mouse (Fc) antibody and anti-human (Fc) antibody. Experiments were performed in duplicate and the error bars denote ± SD, n = 2.