Identification and Characterization of a Novel Linear B-Cell Epitope Within the ASFV pB602L Protein for Serological Diagnosis

African swine fever in both domestic and wild pig populations is caused by the extremely infectious African swine fever virus (ASFV). It seriously endangers biodiversity and results in large financial losses for the worldwide pork sector. The major capsid protein p72 is molecularly chaperoned by the ASFV pB602L protein, which is essential to viral assembly. Furthermore, as a nonstructural protein expressed at late stages of infection, pB602L induces a distinct antibody response that may complement existing serological assays based on structural proteins. Given its strong immunogenicity, pB602L represents a promising antigen for developing supplementary diagnostic tools for African swine fever (ASF). In this study, we successfully generated and separated the ASFV pB602L protein, and we verified its responsiveness using serum from pigs infected with ASFV. Additionally, we produced four monoclonal antibody-specific hybridoma cell lines that targeted the pB602L protein exclusively. These cell lines demonstrated high immunoreactivity and responsiveness toward ASFV pB602L. These results highlight the potential enhancement of diagnostic skills. We have detected two previously unknown linear B-cell epitopes (138TIDSFL143 and 164TNVDTC169) using overlapping peptide and truncated protein fragment analysis. Due to their high degree of conservation across various ASFV strains, these epitopes offer trustworthy candidates for the creation of particular diagnostic instruments. This study expands the known ASFV antigenic repertoire by systematically mapping immunodominant epitopes of pB602L. The identified epitopes provide potential molecular targets for the rational design of multi-epitope subunit vaccines.