Development of a core streptavidin-bridging amplified ELISA based on p54 epitope for high-sensitivity detection of african swine fever virus antibodies.

Infection with the African swine fever virus (ASFV) causes a highly acute and lethal disease in pigs, emphasizing the urgent need for early detection to manage outbreaks effectively. While indirect enzyme-linked immunosorbent assays (ELISAs) based on antigenic epitopes provide high sensitivity, conventional adsorption of epitopes onto ELISA plates can hinder antibody recognition. In this study, we developed an indirect ELISA for ASFV antibody detection by integrating the identified p54 antigenic epitope into a core-streptavidin (cSA)-based immobilization system. Using the 6G9 monoclonal antibody (mAb), generated against the recombinant p54 protein (GST-rp54), we confirmed its specific recognition of ASFV-positive serum. A highly conserved linear epitope within p54, spanning amino acids 60-79 (A60AIEEEDIQFINPYQDQQQWV79; p5460-79), was identified as the 6G9 mAb binding region. Immobilization of biotin-PEG4-p5460-79 via cSA significantly enhanced immunoreactivity, increasing the OD₄₅₀ value by 0.22 (1:800 dilution) and 0.28 (1:400 dilution) for the 6G9 mAb and ASFV-positive serum, respectively. After optimizing key assay parameters, we established a cSA-based indirect ELISA using the p5460-79 epitope (cSA-p5460-79-inELISA). The assay exhibited high reproducibility (coefficient of variation 3.29-8.76 %) and strong specificity, showing no cross-reactivity with antisera against CSFV, PCV2, PRRSV, PRV, PEDV, TGEV, or PDCoV. It also demonstrated superior sensitivity, detecting ASFV-positive serum diluted up to 1:1600 (OD₄₅₀ > 0.43), outperforming a commercial ELISA kit (limit 1:400). Receiver operating characteristic (ROC) analysis revealed 95.5 % sensitivity and 96.2 % specificity based on samples of known status (n = 100). In conclusion, we developed a novel, specific, and highly sensitive serological method for ASF detection, providing an effective tool for early diagnosis.