Development and characterization of monoclonal antibodies against ASFV-encoded early protein pI73R

African Swine Fever Virus (ASFV), a highly pathogenic and lethal large DNA virus, encodes a multitude of viral proteins. Among them, the pI73R is expressed at the very early stages of viral infection and exhibits strong antigenicity, making it a promising ASF diagnostic target. In this study, we successfully expressed the pI73R using an E. coli expression system. After immunizing BALB/c mice, performing cell fusion, and conducting subcloning screening, we obtained two monoclonal cell lines, designated 5D5 and 12F2, capable of stably secreting antibodies against pI73R. Western blot (WB), immunoprecipitation (IP), and indirect immunofluorescence (IFA) confirmed the functional activity of these two monoclonal antibodies (mAbs). The results demonstrated that both mAbs reacted with ASFV during the aforementioned assays. Epitope mapping with truncated pI73R proteins showed that these mAbs can specifically recognize the linear epitope ²¹LTAKNIKVVI³⁰, which is highly conserved across different ASFV genotypes and is localized on the helix α2 of pI73R. Additionally, blocking ELISA and neutralization assays indicated that pI73R-5D5 exhibited limited blocking effects on ASFV infection. In conclusion, monoclonal antibodies targeting pI73R exhibit excellent reactivity and high detection efficiency. These findings highlight their potential for ASFV diagnostics and vaccine development while laying the groundwork for further protein functional studies.