African swine fever (ASF) is a highly lethal hemorrhagic viral disease of domestic pigs caused by African swine fever virus (ASFV). Although a good advance has been made to understand the virus, a safe and effective vaccine against ASFV is still lacking and its eradication solely depends on its early and accurate diagnosis. Thus, improving the available diagnostic assays and adding some validated techniques are useful for a range of serological investigations. The aim of this study was to produce and characterize p54 monoclonal antibodies with an ultimate goal of developing a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for ASFV antibody detection. Five monoclonal antibodies against p54 protein expressed in Escherichia coli was generated and their characterizations were investigated. Furthermore, a competitive enzyme-linked immunosorbent assay (cELISA) based on a monoclonal antibody designated as 2A7 was developed. To evaluate the performance of the assay, a total of 365 pig serum samples (178 negative and 187 positive samples) were tested and a receiver-operating characteristic (ROC) analysis was applied to determine the cut-off value. Based on the ROC analysis, the area under the curve (AUC) was 0.982 (95% confidence interval: 96.9% to 99.4%), besides a sensitivity of 92.5% and a specificity of 98.9% was achieved when the percent inhibition of 20% was selected as a threshold. Moreover, the result showed an excellent agreement when compared to other commercially available blocking ELISA (kappa value = 0.912) and showed no reaction to other swine pathogens. Overall, the newly developed cELISA method offers a promising approach for a rapid and convenient ASFV serodiagnosis, which could be used as alternative to other serological assays for screening possible ASFV infection.

African swine fever (ASF) is a highly contagious hemorrhagic disease of pigs, posing a significant threat to the world pig industry. Several researchers are investigating the possibilities for developing a safe and efficient vaccine against ASF. In this regard, significant progress has been made and some gene-deleted ASFVs are reported as potential live attenuated vaccines. A seven-gene-deleted live attenuated vaccine candidate HLJ/18-7GD (among which CD2v is included) has been developed in our laboratory and reported to be safe and protective, and it is expected to be commercialized in the near future. There is an urgent need for developing a diagnostic method that can clearly discriminate between wild-type-ASFV-infected and vaccinated animals (DIVA). In the present study, a dual indirect ELISA based on p54 and CD2v proteins was successfully established to specifically distinguish serum antibodies from pigs infected with wild-type ASFV or possessing vaccine immunization. To evaluate the performance of the assay, a total of 433 serum samples from four groups of pigs experimentally infected with the wild-type HLJ/18 ASFV, immunized with the HLJ/18-7GD vaccine candidate, infected with the new lower virulent variant, and specific-pathogen-free pigs were used. Our results showed that the positive rate of immunized serum was 96.54% (p54) and 2.83% (CD2v), and the positive rate of the infection by wild-type virus was 100% (p54) and 97.8% (CD2v). Similarly, the positive rate to infection by the new low-virulent ASFV variant in China was 100% (p54) and 0% (CD2v), indicating the technique was also able to distinguish antibodies from wild-type and the new low-virulent ASFV variant in China. Moreover, no cross-reaction was observed in immune sera from other swine pathogens, such as CSFV, PEDV, PRRSV, HP-PRRSV, PCV2, and PrV. Overall, the developed dual indirect ELISA exhibited high diagnostic sensitivity, specificity, and repeatability and will provide a new approach to differentiate serum antibodies between wild virulent and CD2v-unexpressed ASFV infection, which will play a great role in serological diagnosis and epidemiological monitoring of ASF in the future.