The capsid protein p72 specific mAb and the corresponding novel epitope based ELISAs for detection of ASFV infection

The African Swine Fever Virus (ASFV) poses a significant threat to the global pig farming industry. The major icosahedral capsid protein p72 plays a key role in the assembly of virus particles and infection process. As one major antigen of ASFV, p72 has been widely utilized as a marker for diagnosing infection. In this study, five p72 specific monoclonal antibodies (mAbs) were generated through the immunization of mice followed by cell fusion. Among the five hybridomas, clones 1B7, 2F3, 5D3, and 5D4 recognized a novel epitope of ¹⁰¹FHDMVGHHILGACH¹¹⁴, while clone 1D7 recognized a new epitope, ²³⁹GPLLCNIHDL²⁴⁸. Both linear epitopes were found to be highly conserved across all genotypes I and II ASFV isolates, with the first located at the pseudo hexagonal base of the p72 trimer and the latter situated at the FG_N insertion loop. The antigenic epitopes were capable of competing with the binding of corresponding mAbs in p72 protein based ELISA, and peptide based ELISA effectively detected ASFV antibodies in clinical samples. Furthermore, we developed and optimized a sandwich ELISA using the mAb 2F3 for efficient detection of ASFV p72 antigen. Our study not only provides valuable tools for assessing p72 function assay, but also lays the foundation for serological diagnosis, prevention and control of ASF.