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 “ 116 TSSFETLFEQ 125 ,” 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.