Discovery of a Structurally Stable Immunodominant Region in ASFV p30 C-Terminus Using a Panel of Monoclonal Antibodies.

African swine fever (ASF) is a lethal hemorrhagic viral disease of pigs with devastating global socioeconomic impacts. Due to the absence of a safe and effective vaccine, early surveillance and precise diagnostics are critical for ASF prevention and control. The ASF virus (ASFV) p30 protein, one of the most immunogenic early-expressed antigens during infection, is a prime target for diagnostic assays and subunit vaccines. Its comprehensive antigenic characterization remains crucial for rational vaccine design. In this study, we generated a panel of 20 monoclonal antibodies (MAbs) against the ASFV p30 protein, classifying them into 11 groups based on epitope specificity. MAbs from six groups (subgroups I-1, I-2, and II-1, as well as groups III, IV, and V, collectively comprising 15 MAbs) recognized six distinct epitopes within the C-terminus of p30 (137-194 aa), while MAb 16-5E7E8 (subgroup II-2) recognized a distinct epitope within residues 154-190. The p30 C-terminus (137-194 aa) demonstrated structural stability under denaturing conditions (SDS-PAGE and western blot [WB]) and was identified as an immunodominant region through its reactivity with ASFV-positive sera. Notably, MAbs targeting this immunodominant region exhibited modest neutralizing activity, whereas those binding to other regions showed no neutralizing activity. Furthermore, MAb Q4-1F10B7 (group VI) recognized a linear epitope "116TSSFETLFEQ125," MAbs Q1-1G4B2 (group VIII) and Q8-4F9F3 (group IX) recognized conformation-dependent epitopes, and MAb 5-1C8B6 (group VII) likely recognized an aberrant or non-native form of the p30 protein. These results expand the epitope landscape of p30 protein and lay a foundation for ASF diagnosis and vaccine research.