Ferritin nanoparticle-based indirect ELISA for immunodominant region screening and B-cell epitope validation of African swine fever virus.

African swine fever virus (ASFV) poses a severe threat to the global pig industry, creating an urgent need for effective vaccines and reliable diagnostic tools. While numerous ASFV B-cell epitopes have been reported based on murine-derived monoclonal antibodies (mAbs), such epitopes may not fully represent the antibody targets recognized in naturally infected pigs. Therefore, we need an approach to screen immunodominant regions by evaluating the reactivity of pre-defined epitopes with ASFV-infected pigs' sera. In this study, we developed an indirect enzyme-linked immunosorbent assay (iELISA) based on recombinant epitope-ferritin nanoparticles (EFNs), as a platform for immunodominant region screening and epitope validation. This assay, hereafter referred to as EFNs-iELISA, shows an approximately twofold improvement in sensitivity compared with synthetic peptide-based ELISA for linear B-cell epitopes, and improved discrimination over GST-His₆-peptide and BSA-conjugated peptide formats. Using EFNs-iELISA, we evaluated 22 known murine-derived B-cell epitopes and 17 predicted epitopes from ASFV proteins CD2v, P30, P54, and P72 with ASFV-positive and -negative porcine sera. According to the calculated cut-off threshold, tested epitopes were classified into different groups based on positive or negative reactivity. Our analysis confirmed that among 22 known murine-derived ASFV B-cell epitopes, 16 (72%) also showed positive reactions with porcine-derived sera. Moreover, we also screened 7 new porcine-specific immunoreactive regions on these proteins. The EFNs-iELISA method provides a practical tool for screening immunodominant B-cell regions of ASFV, with potential applications in vaccine and diagnostic research.