Abstract

Rapid detection of diverse Vibrio parahaemolyticus serotypes in seafood requires simple and reliable immunoassays. Our prior surface proteomic screening identified outer membrane protein A (OmpA) as a highly abundant and accessible surface antigen. Herein, monoclonal antibodies (mAbs) against OmpA (VPA1186) were generated and applied to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and a lateral flow immunoassay (LFIA). Among the 12 mAbs, 6D11 and 12B1 showed superior binding affinity (3.11-8.17 × 10 9 ) and higher positive-to-negative ratios (>20) when used as mAb pair. Notably, the capture mAb 6D11 showed high specificity for V. parahaemolyticus , whereas 12B1 exhibited broader cross-reactivity within the Vibrio Spp.; the Buckets effect of the mAb pair shapes the high specificity of the methods. The optimized DAS-ELISA achieved a lowest limit of detections (LODs) of 1.37 × 10 4  CFU/mL across multiple V. parahaemolyticus serotypes (6/6) and strains (9/9), while LFIA exhibited a visual LOD of approximately 10 5  CFU/g in spiked sample and 10 CFU/g after a 6-h enrichment. The ELISA and LFIA showed high specificity (93.10%, 27/29 strains) for V. parahaemolyticus . In real seafood samples analysis, this method showed high consistency (95.5%, 21/22 samples) with PCR and culture-based method. By targeting the surface-exposed, abundant, and conserved OmpA antigen, this study established an antigen-defined strategy for specific cross-serotype detection of V. parahaemolyticus in large aquatic products.