Identification of three novel overlapping epitopes within the immunodominant region of ASFV p30 protein facilitates development of a competitive ELISA for antibody detection.

African swine fever virus (ASFV) causes devastating disease in swine. The ongoing global pandemic, driven primarily by genotype II strains and increasingly complicated by the emergence of attenuated mutants and inter-genotypic recombinants, necessitates improved diagnostic tools and deeper understanding of viral antigenic structure. The viral phosphoprotein p30 is an immunodominant antigen expressed early during infection, making it an ideal target for diagnostics and vaccine development. In this study, three novel monoclonal antibodies (mAbs) against p30, designated 2A8 (IgG2b, κ), 2B3 (IgG2a, κ), and 13A7 (IgG1, κ), were generated, all exhibiting strong reactivity with recombinant and native p30 in ASFV-infected macrophages. Epitope mapping identified three precise linear overlapping epitopes within the immunodominant aa 76-93 region: 76EHQAQEEWNM85 (2A8), 83WNMILHVLFEE93 (2B3), and 76EHQAQEEWNMILHVLF91 (13A7), all completely conserved across genotypes I, II, and I/II recombinants. Competitive binding assays revealed mutual inhibition among the mAbs consistent with epitope overlap, with mAb 13A7 demonstrating the strongest competition with a representative ASFV-positive swine serum. Leveraging this competitive inhibition profile, a competitive ELISA was developed for ASFV antibody detection. The assay exhibited satisfactory analytical sensitivity and showed no cross-reactivity with the sera positive for other major porcine viruses, and achieved 97.38% agreement (κ = 0.941) with a commercial p72 mAb-based competitive ELISA kit when testing 344 field sera. This study identifies aa 76-93 as a highly antigenic region within p30 and provides a reliable tool for ASFV antibody detection with significant potential to enhance surveillance and control programs amid the evolving complexity of the ASFV pandemic.