Indirect ELISA Based on ASFV Polymerase Three Subunits for Serological Monitoring of African Swine Fever Antibodies

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a highly contagious and fatal disease. Accurate detection in the early stages of an outbreak relies on molecular methods, but serological monitoring at the population level is also crucial for assessing the extent of exposure and past infections. This experiment developed an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against ASFV, using three ASFV RNA polymerase subunits (H359L, C147L, and D339L) as coating antigens. The recombinant proteins were successfully expressed in <i>Escherichia coli</i> and purified. Using a checkerboard titration method, we systematically optimized key assay parameters, determining the optimal coating conditions to be a mixture of H359L, C147L, and D339L at a volume ratio of 1:2:2, with individual concentrations of 1 μg/mL, 0.4 μg/mL, and 0.5 μg/mL, respectively. Other optimized parameters included a serum dilution of 1:200, a blocking buffer containing 5% skim milk, and specific incubation conditions for the secondary antibody and substrate. The cut-off value was established at 0.430 (x¯ + 4SD) based on 30 negative sera. The established triple-antigen indirect ELISA exhibited high sensitivity (detecting positives at dilutions up to 1:3200) and excellent specificity (no cross-reactivity with antisera against CSFV, PRRSV, PRV, PCV2, and PEDV. Both intra and inter assay repeatability were confirmed, with coefficients of variation ranging from 1.020% to 7.600%. Validation with 123 clinical serum samples demonstrated a 96.75% concordance rate with a commercial kit. In conclusion, the three-antigen indirect ELISA established in this study exhibits high specificity and sensitivity, making it suitable for serological surveillance and exposure assessment of ASFV antibodies. It can be combined with molecular detection for epidemiological investigations and integrated prevention and control measures.