A triple protein-based ELISA for differential detection of ASFV antibodies

African swine fever (ASF) caused by the ASF virus (ASFV) is a severe and highly contagious viral disease that poses a significant threat to the global pig industry. As no vaccines or effective drugs are available to aid prevention and control, early detection is crucial. The emergence of the low-virulence ASFV strain not expressing CD2v/MGFs (ASFVΔCD2v/ΔMGFs) has been identified domestically and internationally and has even become an epidemic in China, resulting in a complex epidemic. The commercialized ASFV ELISA kits available can detect the presence of ASFV infection in pigs, but they are unable to distinguish wild-type ASFV from gene-deleted strains. The current published ELISA assays can distinguish between the wild-type and CD2v gene-deleted ASFV but cannot differentiate wild-type and MGF505 gene-deleted ASFV or CD2v and MGF505 double-gene deleted ASFV infection, posing new challenges for an effective prevention and control of ASFV. In this study, the ASFV-p30, ASFV-CD2v, and ASFV-MGF505 proteins were expressed using a prokaryotic expression system, and a triple protein-based ELISA antibody detection method based on these proteins was successfully established to effectively differentiate between wild-type ASFV and ASFVΔCD2v and/or ASFVΔMGF505 virus infection. This triple protein-based ELISA showed good analytical specificity without cross-reactivity with antibodies against PRRSV, CSFV, PRV, and PCV2. Moreover, it demonstrates remarkable analytical sensitivity by allowing the identification of clinical samples even at dilutions as high as 1:800. The coefficient of variation the intra-assay and inter-assay were below 5%, indicating strong repeatability and reproducibility. To evaluate the performance of the triple protein-based ELISA, a total of 59 clinical serum samples were detected using the triple protein-based ELISA. The results showed that 22 samples were positive for ASFV, of which 19 were ASFV wild-type, one was ASFVΔCD2v, and two were ASFVΔMGF505. Compared with the commercialized triplex qPCR kit, the triple protein-based ELISA exhibited high diagnostic sensitivity and diagnostic specificity. The test accuracy with the commercialized triplex qPCR kit was 98.31% (58/59), and the test accuracy with the commercialized ELISA kit was 96.61% (57/59). These results indicated that the developed triple protein-based ELISA performs well in detection and differentiation. Therefore, it will be useful for the ASFV serological differential diagnosis and epidemiology study.