Development and Preliminary Validation of a Recombinant DP238L-Based Indirect ELISA for the Serological Detection of African Swine Fever Virus Antibodies

African swine fever virus (ASFV) antibody detection is an important component of serological surveillance and retrospective investigations. DP238L was recently identified by proteome-wide serum-antibody profiling as a frequently recognized ASFV antigen, and recombinant DP238L was further shown to react with ASFV-positive sera and to elicit antigen-specific antibody responses in immunized pigs. Here, we extend those observations toward diagnostic assay development and preliminary validation. Full-length DP238L from ASFV strain SY-1 (GenBank OM161110) was evaluated as a single recombinant coating antigen for an indirect ELISA (DP238L-iELISA). Independent sequence analysis showed that SY-1 DP238L contains 238 amino acids, with a theoretical molecular mass of 27.13 kDa, a theoretical pI of 4.92, and a GRAVY value of −0.97. In a quality-restricted subset of complete, unambiguous 238-aa homologs (n = 7), amino acid identity to SY-1 ranged from 97.48% to 100% (median, 98.74%). Recombinant DP238L was expressed and purified from Escherichia coli and specifically recognized by ASFV-positive swine serum. ELISA optimization identified a coating antigen concentration of 0.5 μg/mL, a secondary-antibody working dilution of 1:2500 (0.4 μg/mL), a serum dilution of 1:100, a serum incubation time of 45 min, and a substrate development time of 15 min. Using 518 clinical swine serum samples categorized according to the commercial INgezim PPA COMPAC K3 blocking ELISA, ROC analysis yielded an AUC of 0.9989 (bootstrap 95% CI, 0.9973–0.9999). At the selected S/P cutoff of 0.30, positive percent agreement and negative percent agreement relative to INgezim were 97.2% and 98.7%, respectively; overall agreement was 98.3% and Cohen’s κ was 0.956. A limited six-sample heterologous panel provided preliminary cross-reactivity information, with all tested samples remaining below the cutoff. In a longitudinal panel from five experimentally infected pigs, three animals exceeded the cutoff at 7 days post-infection (dpi) and all five exceeded it by 10 dpi. These findings support DP238L as a promising recombinant antigen for ASFV serological assay development while emphasizing that the present results represent an internal comparator-based preliminary evaluation requiring broader independent validation.