Computational and experimental antibody affinity and diagnostic accuracy quantification of SARS-CoV-2 SD2 major disulfide loop analog

Introduction Synthetic oligopeptides provide a rapid and cost-efficient approach to developing antibodies and diagnostics for emerging viral variants. Methods This study computationally and experimentally characterized a synthetic peptide analog of the SARS-CoV-2 spike subdomain 2 major disulfide loop (SD2MDL), designated S621 (CPVAIHADQLTPTWRVYSTC). Binding affinity was computationally estimated using the Heuristic Affinity Prediction Tool for Immune Complexes (HAPTIC), while experimental validation was performed using enzyme-linked immunosorbent assay (ELISA) with rabbit-derived antipeptide antibodies. Clinical diagnostic accuracy testing was done using plasma samples from RT-PCR–confirmed COVID-19 patients and pre-COVID-19 controls. Results S621 demonstrated nanomolar binding affinity ( K d app = 1.14 nM) and high avidity (3.67 nM), closely matching HAPTIC predictions (3.54 nM). Diagnostic evaluation yielded a sensitivity of 75.14% and specificity of 53.18%, corresponding to an overall accuracy of 71.79%. Discussion These findings demonstrate that a single synthetic peptide derived from a conserved spike subdomain can function as a high-affinity surrogate for full-length antigens, supporting its potential application in rapid peptide-based immunodiagnostics.