Development of an indirect ELISA based on a multi-epitope recombinant protein for antibody detection against African...

African swine fever (ASF) is a highly contagious and fatal hemorrhagic disease of swine. While vaccines have been licensed in Vietnam and the Philippines, their global application remains constrained by insufficient large-scale validation of safety and efficacy, rendering rapid and reliable diagnostic assays crucial for effective prevention and control. In this study, a multi-epitope antigen (MEA) was rationally designed by utilizing informatics to predict and select 15 conserved B-cell epitopes from four major African swine fever virus (ASFV) structural proteins: p30, p54, p72, and pA104R. The recombinant MEA was expressed in Escherichia coli, and its immunogenicity was validated by Western blot and indirect immunofluorescence assays. This antigen was subsequently utilized as the basis for an indirect enzyme-linked immunosorbent assay (iELISA) whose reaction conditions were systematically optimized. The optimal cutoff value for the iELISA was determined to be 0.315 from the analysis of 100 known ASFV-negative serum samples. The assay demonstrated excellent reproducibility, with intra- and inter-assay coefficients of variation below 10%, and exhibited high specificity, with no cross-reactivity against sera positive for other common swine viruses. Notably, the developed assay exhibited high sensitivity, capable of detecting ASFV antibodies in serum samples at dilutions up to 1:6,400. Furthermore, it demonstrated superior sensitivity compared to two commercial ELISA kits in validating clinical samples from regions with circulating Genotype II strains. These findings highlight the successful development of a sensitive and specific iELISA for ASFV antibody detection, representing a valuable new tool for large-scale epidemiological surveillance and the implementation of robust control strategies for ASF.IMPORTANCEAfrican swine fever (ASF) is a devastating viral disease of swine causing substantial economic losses globally. Although vaccines have recently been licensed in countries such as Vietnam and the Philippines, the global availability of a universally standardized and safe vaccine remains a significant challenge. Control efforts continue to rely heavily on rapid and accurate diagnosis. A significant limitation of current serological assays is their variable sensitivity, which can lead to missed detections and compromise surveillance programs. This study addresses this deficiency through a rational, epitope-based engineering approach. We designed a single, chimeric antigen by genetically fusing 15 conserved, immunodominant B-cell epitopes from four major African swine fever virus proteins. The resulting indirect enzyme-linked immunosorbent assay demonstrated high specificity, excellent reproducibility, and, critically, superior diagnostic sensitivity compared to two commercial kits. This work reports the development of a significantly improved serodiagnostic tool, offering enhanced reliability for large-scale epidemiological surveillance and supporting more robust control strategies against ASF.