Fig 2: Validation of full spike proteins (wild type and variants) and ACE2 binders in the serosurveillance of clinical samples.a Molecular modeling of the distances between N-terminus of ACE2 to the N-termini of nearest S protein is ~48 Å and ~88 Å, respectively (PDB ID: 7A97). b Comparison of signal and background for the full spike (wild type and variants) and ACE2 pairs. c Serial dilution of a commercially available NAb (Sino Biological, 40592-R001) in the presence of ß10-(S)WT and ß9-ACE2-Fc pair. d Serum samples that have been tested previously on two different COVID-19 detection assays were also tested using Neu-SATiN. Wild type full spike protein with ß10 tag (ß10-(S)WT) and ACE2 with ß9 tag (ß9-ACE2-Fc) was used as the binders. Signals from patient samples were normalized to the signal from normal human serum with the binders alone (no neutralizing antibodies). The red dotted line at 10% activity (i.e., 90% neutralization) indicates the cutoff line to distinguish neutralizing samples from the non-neutralizing samples; ‘+’ and ‘-‘ signs below the sample number indicate the result from two prior tests detecting anti-SARS-CoV-2 antibodies (COV2G Siemens 1st Gen and EUROIMMUN EIA). e Activity measured in protein-based surrogate neutralization assay (pbSNA) versus neu-SATiN for the wild-type (WT) variant shows high correlation with a Pearson’s correlation test r value of 0.88. N = 66. f Neutralization efficacy of patient samples against WT and variant S proteins. Samples used in (d) (n = 43) were tested with an additional n = 35 patient samples that were positive for anti-SARS-CoV-2 antibodies. Luminescence signals from each patient serum were measured and normalized to human serum (no NAb). Known post-vaccination, post-infection, and negative (no known infection or vaccination) samples were plotted separately. Red dotted line at 10% indicates the cutoff between the negative samples and the positive samples for distinction of neutralization. g Comparison of serum data from patients that were vaccinated to patients that were vaccinated after a documented infection (n = 40). All patient samples were collected before November 2021, prior to any known Omicron infections. Source data are provided as a Source data file.