Identification of a New Conserved Antigenic Epitope by Specific Monoclonal Antibodies Targeting the African Swine Fever Virus Capsid Protein p17

African swine fever (ASF) has widely spread around the world in the last 100 years since its discovery. The African swine fever virus (ASFV) particles are made of more than 150 proteins, with the p17 protein encoded by the D117L gene serving as one of the major capsid proteins and playing a crucial role in the virus’s morphogenesis and immune evasion. Thus, monoclonal antibody (mAb) targeting p17 is important for the research and detection of ASFV infection. Here, we produced two specific mAbs against p17, designated as 1G2 and 6G3, respectively, and both have been successfully used in enzyme-linked immunosorbent assay (ELISA), Western blotting, and immunofluorescence assay. Moreover, we found that both 1G2 and 6G3 mAbs recognize a novel epitope of 72–78 amino acids of p17 protein, highly conserved across all genotype I and II strains. Based on this epitope, an indirect ELISA has been established to effectively detect antibodies during ASFV infection, and it exhibits high consistency with commercial ASFV ELISA kits. In summary, the production of the specific p17 mAbs and the identification of the recognized epitope will significantly promote the serological diagnosis of ASFV.