Epitope mapping of diagnostic antibodies targeting SARS-CoV-2 nucleocapsid protein using hydrogen-deuterium exchange mass spectrometry.

The SARS-CoV-2 nucleocapsid (N) protein is a primary target for diagnostic antibody-based detection of COVID-19. To enhance diagnostic accuracy and resilience against emerging variants, it is essential to map the binding sites of diagnostic antibodies. This study employs hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify the epitopes of diagnostic antibodies targeting the N protein. HDX-MS provides high-resolution insights into protein-antibody interactions by detecting changes in solvent accessibility and protein dynamics upon antibody binding. Our findings revealed distinct epitopes critical for antibody specificity and binding, highlighting conserved regions of the N protein that are crucial for diagnostic applications. This work demonstrates the value of HDX-MS in guiding the design of robust diagnostic tools, emphasizing its role in understanding antibody interactions and adapting diagnostics to future SARS-CoV-2 variants. These findings support the development of next-generation diagnostics with improved sensitivity, specificity, and durability.