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The p54 protein is encoded by the ASFV E183L gene and is an important structural protein located on the inner envelope of the virus. It is involved in processes of virus assembly, apoptosis induction, and neutralizing antibody production. In this study, three specific monoclonal antibodies (mAbs) against ASFV p54 protein were generated, namely 6B11, 3E3, and 3C10, from mice who were immunized with recombinant prokaryotic p54-truncated protein. Three novel linear B cell epitopes, recognized by the mAbs, were revealed: 60AAIEEEDIQFINP72, 128MATGGPAAAPAAASAPAHPAE148, and 163MSAIENLRQRNTY175. The epitopes 60AAIEEEDIQFINP72 and 163MSAIENLRQRNTY175 were highly conserved in genotype I and II ASFV strains. In addition, the epitope peptide ELISA can be used for the detection of ASFV antibodies. Our work provides new insights for p54 antigenicity and an alternative tool for serological diagnosis of ASF.<\/jats:p>","DOI":"10.3390\/ani15091296","type":"journal-article","created":{"date-parts":[[2025,5,1]],"date-time":"2025-05-01T05:10:32Z","timestamp":1746076232000},"page":"1296","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["The Novel Antigenic Epitopes of African Swine Fever Virus Inner Membrane p54 Protein Revealed by Monoclonal Antibodies"],"prefix":"10.3390","volume":"15","author":[{"given":"Jiajia","family":"Zhang","sequence":"first","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety, Yangzhou University, Yangzhou 225009, China"},{"name":"Comparative Medicine Research Institute, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kaili","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety, Yangzhou University, Yangzhou 225009, China"},{"name":"Comparative Medicine Research Institute, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shaohua","family":"Sun","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Joint International Research 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Institute, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mingwang","family":"Xie","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety, Yangzhou University, Yangzhou 225009, China"},{"name":"Comparative Medicine Research Institute, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pingping","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety, Yangzhou University, Yangzhou 225009, China"},{"name":"Comparative Medicine Research Institute, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0939-331X","authenticated-orcid":false,"given":"Wanglong","family":"Zheng","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China"},{"name":"Joint International Research Laboratory of Agriculture and Agri-Product Safety, Yangzhou University, Yangzhou 225009, China"},{"name":"Comparative Medicine Research Institute, Yangzhou University, Yangzhou 225009, China"},{"name":"Jiangsu 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