Development and characterization of monoclonal antibodies against p72 protein of African swine fever virus reveals a novel conserved B-cell epitope

• Discovery of a novel and conserved linear B-cell epitope (aa 130-152) on the major capsid protein p72 of ASFV. • Structural insights confirm the epitope's surface accessibility on the p72 trimer, making it a prime target for antibodies. • Validated utility for serodiagnosis through strong reactivity with sera from infected pigs. African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious disease that has spread globally, posing a significant threat to swine production and international trade. As rapid diagnosis is crucial for controlling ASF, its major capsid protein, p72, has become a key target for diagnostic and vaccine development. In this study, we generated five monoclonal antibodies (mAbs) against thep72 protein by immunizing mice with inactivated virus. Using phage display technology, we identified the epitope for one mAb as a novel linear B-cell epitope within amino acids 130-152 of the p72 protein. Structural and homology analyses revealed that this epitope is highly conserved across diverse ASFV genotypes and is exposed on the surface of the p72 trimer. Importantly, the epitope showed strong reactivity with sera from ASFV-positive swine. These findings offer a foundation for creating improved serological diagnostics and designing epitope-based vaccines against ASFV.