Establishment of an immunoperoxidase monolayer assay for the detection of African swine fever virus antibodies

African swine fever (ASF) is a lethal infectious disease affecting domestic and wild pigs, caused by the African swine fever virus (ASFV), with a mortality rate of up to 100 %. The evolving prevalence and variation of ASFV has led to the emergence of low-virulence strains, which induced chronic infections and posed challenges in nucleic acid-based diagnostics due to potential false negatives. This underscores the urgent need for reliable antibody monitoring to facilitate early diagnosis. In this study, based on a highly attenuated BK2258 cell-adapted strain HLJ18/BK33, we established an immunoperoxidase monolayer assay (IPMA) for ASFV antibodies detection. After optimization using a total of 608 pig sera, the performance of the assay was better than that of the commercial iELISA with higher sensitivity and specificity. The newly established IPMA method demonstrated high specificity with no cross-reactivity with positive sera for six other important porcine pathogens. The IPMA method developed in the present study could serve as the potential gold standard for serological diagnosis and evaluation of other detection methods for ASFV antibodies, owing to its high sensitivity and specificity. Furthermore, the IPMA method will provide a new and effective strategy for ASF monitoring, prevention and control in China.