A Novel In-Cell ELISA With Superior Sensitivity and Specificity for the Detection of African Swine Fever Virus-Specific IgM and IgG Antibodies.

African swine fever (ASF), a high-profile transboundary animal disease caused by ASF virus (ASFV), imposes a devastating impact on the global swine industry. Given that vaccines are still under development, including field evaluations, early detection of ASFV is crucial for effective disease control and mitigation. Although PCR is the primary viral detection method of acute or subacute ASFV infections, antibody detection plays a unique role in detecting low-virulent ASFV infection, identifying recovered animals, and tracking viral transmission. ELISA for ASFV antibody detection is commonly used for initial serological screening. To avoid false positive results, the World Organisation for Animal Health (WOAH) recommends using a second serologic method, such as the indirect immunofluorescence assay (IFA), indirect immunoperoxidase test (IPT), or immunoblot test, to confirm the ELISA-positive cases. This strategy improves specificity but not sensitivity (i.e., false negative cases persist). To address this issue, a novel in-cell ELISA (icELISA) was developed in this study. Receiver operating curve analysis of the icELISA revealed the optimized cutoff value of sample-to-positive ratio (S/P ratio) was at 47% with 99.46% analytical sensitivity and 99.43% analytical specificity. Results of the comparative diagnostic sensitivity analysis showed that positive detections of icELISA (150 samples) surpassed a blocking ELISA-IPT combination (132 samples) by 18 samples. Further investigation revealed that the 18 samples contained ASFV-specific immunoglobulin M (IgM) antibodies instead of immunoglobulin G (IgG). The results suggested the icELISA can detect both ASFV-specific IgG and IgM, which outperforms a blocking ELISA-IPT combination in earlier detection, particularly when only IgM antibody is present in a test sample.