Development of a native-antigen Western blot using ZZ-R 127–derived FMDV non-structural proteins for qualitative serology to inform DIVA workflows

This study describes the development of a Western blot (WB) assay employing native non-structural proteins (NSPs) from foot-and-mouth disease virus (FMDV)-infected ZZ-R 127 cell lysates to support confirmatory serodiagnosis within DIVA (differentiation of infected from vaccinated animals) frameworks. Native antigens were generated from six FMDV isolates representing serotypes A, O, and Asia-1 and were separated by denaturing SDS-PAGE. Antigen identity was verified by co-migration with recombinant counterparts and detection with monoclonal antibodies (MAbs). Using one NSP-positive and one NSP-negative polyclonal bovine serum, the assay demonstrated consistent identification of 2C, 3D, and an ~25.8kDa 3AB-consistent precursor as the most reliable diagnostic targets. While MAb profiling confirmed the presence of 3C in the lysates, it showed no reactivity with the polyclonal serum, indicating limited utility for serology. Pixel-based quantification revealed predictable dilution-dependent signal behavior within gels (R2 > 0.91 on log-log plots), though inter-gel variability was substantial, favoring qualitative over quantitative application. Absence of signals in mock-infected cell lysates and in the negative serum supported specificity. By preserving the native processing context of immunodominant NSPs, this platform is well-suited as a second-tier tool to adjudicate equivocal ELISA results in vaccinated populations.