Background: The N-terminal ectodomain (NTE) of the SARS-CoV-2 membrane (M) glycoprotein is a short, flexible region that remains exposed on the virion surface. Despite its small size and conformational plasticity, this region contains conserved linear epitopes that may serve as practical surrogates for full-length proteins in serological diagnostics.
Objective: The objective of this study was to develop and evaluate a synthetic peptide-based diagnostic assay targeting the NTE of the SARS-CoV-2 M protein.
Methods: Peptide analogs of SARS-CoV-2 M NTE were designed from a cluster of epitopes retrieved on February 2021 from the Immune Epitope Database (IEDB). M1 (CADSNGTITVEELKKLLEQC-am) is a polymerizable peptide used as coating antigen in a diagnostic indirect ELISA, while anti-M1 is an antipeptide antibody affinity-purified from the sera of rabbits immunized with M1i (MADSNGTITVEELKKLLEQC-am). The binding affinity of monomeric and polymeric M1 was estimated in terms of the dissociation constant (Kd), taken from the slope of ELISA data performed against anti-M1, and linearized using the Liliom method. To determine diagnostic accuracy, M1-based indirect ELISA was performed on 221 biobanked plasma controls and on 1,222 serum samples corresponding to 544 RT-PCR–confirmed adult patients admitted to the Philippine General Hospital, a 1,335-bed tertiary referral center, during October 2020–February 2021, before SARS-CoV-2 vaccines became available locally.
Results: Polymeric M1 exhibited a two-fold gain in apparent affinity (Kd = 4.33 nM) compared with the monomeric form (Kd = 8.00 nM). Clinical validation of the M1-based diagnostic ELISA yielded a post hoc (Youden J) sensitivity of 95.26% and specificity of 52.27%, with an overall diagnostic accuracy of 88.70%.
Conclusion: M1 demonstrates that synthetic peptide antigens can serve as stable, high-sensitivity antigens for diagnostic assays. This design principle may be applied to other emerging pathogens where rapid assay development and scalability are critical.
