Salivary agglutinin causes glycan-mediated interference in ELISA-based antigen detection

Saliva represents an attractive alternative diagnostic matrix for viral antigen detection due to its non-invasive collection and ease of use. However, a consistent reduction in the diagnostic sensitivity of saliva-based rapid antigen detection tests (RADTs) has been reported, and the biochemical factors underlying this reduced performance remain incompletely understood. In this study, we investigated the role of salivary agglutinin (SAG), a highly glycosylated innate immune protein abundantly present in saliva, as a source of matrix-dependent interference in ELISA-based detection of the SARS-CoV-2 spike (S) protein Using in-house ELISAs and a commercial protein S ELISA kit, we demonstrate that SAG binds both the antigen and monoclonal anti-S IgG antibodies used as capture and detection reagents. These interactions occur at a concentration of SAG within the reported physiological range in saliva and result in increased background signal and impaired assay performance. Given that SAG interacts with glycoproteins, capture and detection antibodies were enzymatically deglycosylated using Endo S to assess whether SAG interference could be mitigated. Deglycosylation markedly reduced SAG binding to detection antibodies and to capture antibodies prior to surface immobilization, indicating that SAG-IgG interactions are influenced by antibody glycans. Overall, these findings identify SAG as a relevant contributor of matrix-dependent analytical interference in saliva-based ELISA assays and highlight antibody deglycosylation as a potential pretreatment approach to improve the performances of saliva-based RADTs.