Development and Evaluation of Three Indirect ELISA Methods Based on Novel Recombinant ASFV Antigens for Serological Detection of African Swine Fever.

African swine fever (ASF) poses a catastrophic and ongoing threat to the global swine industry. The rapid intercontinental spread of ASF, compounded by the absence of licensed vaccines and the structural complexity of African swine fever virus (ASFV), renders disease control and eradication extraordinarily challenging. Serological diagnosis is central to long-term ASF surveillance and control, with current assays predominantly based on well-validated antigens such as p30 (CP204L), p54 (E183L), and p72 (B646L), which form the backbone of OIE/WOAH-recommended testing. To expand and complement this established diagnostic repertoire with additional, less-explored targets. In this study, three ASFV proteins, B169L, A151R, and KP177R, were identified through systematic bioinformatic screening and successfully expressed using a prokaryotic expression system. Three individual indirect ELISA methods were subsequently established, optimized, and comprehensively evaluated. All three assays demonstrated high specificity, with no cross-reactivity detected against antisera to PRRSV, CSFV, or PCV2. Intra-assay coefficients of variation (CVs) were below 6% and inter-assay CVs were below 8%, confirming excellent repeatability and reproducibility. These assays provide sensitive, specific, and practically deployable tools that expand the available options for ASF serological surveillance and may serve as useful complements to existing diagnostic methods in epidemiological investigations of this devastating disease.