A double-antibody sandwich enzyme-linked immunosorbent assay with novel anti-VP1 monoclonal antibodies for the detection of porcine Sapelovirus.

Porcine sapelovirus (PSV) is an important porcine pathogen that causes diseases such as diarrhea, encephalomyelitis, pneumonia, and reproductive disorders. In this study, we generated three monoclonal antibodies (mAbs 3A1, 1E6, and 2E4) targeting the PSV VP1 capsid protein using the hybridoma technique. All of them were characterized as IgG1 isotype for the heavy chain and kappa (κ) isotype for the light chain. The minimal B-cell epitope units recognized by 3A1, 1E6, and 2E4 are 87EDTLDYTN94, 44AAETGE49, and 33MINQGD38, respectively. Through systematic antibody pairing screening, we developed a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using mAb 2E4 as the capture antibody and horseradish peroxidase (HRP)-conjugated mAb 3A1 as the detection antibody. The cut-off value was determined to be 0.344, and it exhibited both high sensitivity and specificity, with a detection limit of 0.2 log10 50% tissue culture infective dose per 0.1 mL (TCID50/0.1 mL). This detection method demonstrated a favorable concordance with quantitative PCR (qPCR) and is well-suited for rapid on-site testing. Collectively, these findings establish a foundation for understanding the PSV VP1 protein, guiding rational vaccine design, and developing sensitive diagnostic methods.