Diagnostic Accuracy of an Immunoassay Using Avidity-Enhanced Polymeric Peptides for SARS-CoV-2 Antibody Detection

ABSTRACT Background There is a need for synthetic peptide-based serologic assays that exploit avidity to replace whole antigens while enabling low-cost diagnostics in resource-limited settings. Objective To evaluate the diagnostic accuracy of a polymeric peptide-based ELISA leveraging avidity to enhance signal. Method A 15-member SARS-CoV-2 peptide library corresponding to multiple epitope clusters and proteins was screened by indirect ELISA using pooled sera from RT-PCR-confirmed COVID-19 patients to identify peptides with possible diagnostic utility. The identified lead candidate, S559, possessed terminal cysteine-substitution to allow disulfide polymerization, and the resulting avidity gain was evaluated by comparing the apparent dissociation constant (K D app ) before and after depolymerization with N-acetylcysteine. The performance of an optimized ELISA using S559 was evaluated on 1,222 prospectively collected COVID-19 serum samples and 218 biobanked pre-COVID control serum samples. Results Polymeric S559 with a K D app of 29.26 nM -1 was demonstrated to have a 218% avidity gain relative to the completely depolymerized form. At pre-defined thresholds, the optimized S559 ELISA has a sensitivity and specificity of 83.39% (95%CI: 81.18% and 85.43%) and 96.79% (95%CI: 93.50% and 98.70%), respectively. At post hoc thresholds determined by Youden index, sensitivity and specificity reached 95.01 (95% CI: 93.63% - 96.16%) and 100.00% (95% CI: 98.32% - 100.00%), respectively. Conclusion Homomultivalent epitope presentation using polymeric S559 allows a highly specific immunoassay using human sera that may have important value in detecting antibodies, whether for diagnosing infection, confirming vaccination status or conducting surveillance.