Development and Standardization of Indirect ELISA for African Swine Fever Virus Using Recombinant p30 Protein Produced in Prokaryotic System

Abstract

African Swine Fever (ASF), caused by the African Swine Fever Virus (ASFV), is a highly contagious hemorrhagic disease with high mortality (≈100%) in pigs and is considered the most devastating disease to date. Given the importance of this disease, we aimed to assess the use of the recombinant p30 protein as the sole antigen for the development of an accurate and precise ELISA test (iELISA) for the virus. The recombinant p30 protein (rp30) was produced in a bacterial expression system using a SUMO-tagged expression vector. Protein expression was confirmed by Western blot analysis and purified using affinity chromatography. Antigenicity was evaluated in CF-1 mice, which demonstrated the ability to generate high levels of specific antibodies. The rp30 showed a sensitivity of 95.6% when used in the development of iELISA, a specificity of 92.3%, and a kappa index (κ) of 0.836. Furthermore, reference sera (OIE-ASF) were used to validate the assays, and the results demonstrated an excellent capacity to detect ASF antibodies using only the rp30 antigen up to a serum dilution of 1:100. The inter- and intra-assay variability coefficients were 4.27% and 4.85%, respectively, demonstrating that the assay was accurate and reproducible, allowing its use in seroepidemiological analyses for ASF surveillance.