His tag

rSARS-CoV-2 spike protein was immobilized onto the surface of 96-well microtiter plates by direct adsorption at 2°C to 8°C, followed by washing and blocking. Serial dilutions of human serum samples, including assay quality controls (QCs), were then added to the spike-coated wells and any molecules that could bind to the spike protein, presumptively primarily spike-specific antibodies, were allowed to complex with the immobilized spike protein (for 1 hour at 24°C ± 2°C). After a plate-washing step, a fixed concentration of human ACE2 receptor (hACE2) with a polyhistidine tag (His-Tag) (SinoBiological) was added to the plate for incubation (1 hour at 24°C ± 2°C) during which the hACE2 bound to the spike protein residues with binding sites not obstructed by bound antibody. After washing, the hACE2 receptor bound to the spike protein was then detected using a mouse anti–His-Tag HRP conjugate (Southern Biotech) and a colorimetric signal generated by the addition of TMB substrate. The amount of bound hACE2 detected was inversely proportional to the amount hACE2 binding inhibitors (antibodies) in human serum; inhibitory activity is reported as 50% inhibitory titers based on a 4-PL curve fit (SoftMax Pro software).

EGF (Sigma–Aldrich, SRP3027; 0.1 μg/μL), HGF (Peprotech, 100-39H; 0.1 μg/μL), PDGF-B (Peprotech, 100-14B; 0.1 μg/μL), SARS-CoV-2 RBD (mFc tag, Sino Biological, 40592-V05H; 0.5 μg/μL), porcine insulin (Aladdin, 12584-58-6; 0.5 μg/μL), or PLAU (His tag, Sino Biological, 10815-H08H-A; 0.2 μg/μL) was mixed with probe 1 (final concentration of 0.16 μg/μL) at a protein-to-probe mass ratio of 1:2 in 50 mM HEPES (pH 8.2) for 10 min at room temperature (RT). The required amount of ligand for three replicates was labeled at once, and aliquoted for each replicate. The ligands required for three replicates were labeled at once and divided into equal amounts for each replicate. Equal amount of BSA (Sangon Biotech), glycine or Tris was used as control. The reaction was quenched by adding glycine or Tris buffer (pH 6.8). The N-deglycosylated PLAU was prepared by incubation of PLAU with 24 units/μL peptide-N-glycosidase F (PNGase F, New England Biolabs, P0704S) in 50 mM HEPES (pH 8.2) for 1 h at 37 °C.