Cpass kit

The following proteins were purchased from Sino Biological (Beijing, China): SARS-CoV-2 recombinant Spike RBD wild type, RBD (S477N), RBD (Y453F), RBD (K458R), RBD (F342L), RBD (V367F), RBD (N354D), RBD (A435S), RBD (V483A), RBD (W436R), RBD (G476S), RBD (R408I), RBD (K417N), RBD (Y505C), RBD (N501Y), and human ACE2-histag. The different anti-SARS-CoV-2 Spike protein antibodies were purchased from Sino Biological, Active Motif (Carlsbad, USA), Biolegend (San Diego, CA), ACRO Biosystems (Newark, USA), and Absolute Antibody (Oxford, United Kingdom) (Table 1). HRP conjugated anti-human IgG was from Southern Biotech (Birmingham, AL, USA). Human ACE2-derived peptides described by Cao et al.^{9 (link)} were custom ordered from Peptide 2.0 and the list is presented in Supplementary Table 1.
Lumit SARS-CoV-2 Spike RBD:ACE2 immunoassay components were from Promega (Madison, USA) and they consist of 0.5 μM SARS-CoV-2 RBD-rabbit Fc (RBD-Fc), 0.5 μM human ACE2-mouse Fc (ACE2-Fc), Lumit Detection Substrate C, 10X Lumit Immunoassay Buffer C, Lumit anti-rabbit Ab-SmBiT and Lumit anti-mouse Ab-LgBiT. The Lumit anti-rabbit Ab-LgBiT and Lumit anti-mouse Ab-SmBiT used in some control experiments were also from Promega. c-Pass kit was purchased from Genscript (Piscataway, New Jersey).

sVNT assay is an assay that assesses the inhibition of viral RBD binding to the human receptor, hACE2, and, therefore, is used as a proxy for virus neutralization [26 (link)]. We measured the inhibition of RBD binding to hACE2 using the commercial cPASS kit (Genscript, Singapore) as per manufacturer guidelines with a minor change in the serum dilution. Briefly, mouse sera were diluted 1:2,000 in sample dilution buffer, then incubated with diluted HRP conjugated RBD for 30 minutes at 37 °C. Mixture is then added to ELISA plates coated with hACE2 and incubated for 15 minutes at 37 °C.

For the ACE2 competition ELISA, the cPass kit from GenScript was used (catalog no. L00847). Manufacturer’s instructions were followed. In short, serially diluted sera were incubated with SARS-CoV-2 Spike RBD-HRP and added to ACE2 precoated plates. Plates were developed with 3,3′,5,5′-tetramethytlbenzidine for 10 min at RT, and the reaction was stopped with 1 N sulfuric acid. The absorbance at 450 nm was measured by an ELISA plate reader. Titers were determined at an absorbance cutoff of 0.5 OD.