Paper-based immunosensor for quantitative detection of African swine fever virus p54 protein

African swine fever virus (ASFV) has resulted in significant economic detriment to the livestock industry in recent years. Highly sensitive and accurate detection methods are currently the most effective means for ASFV prevention and control. In this work, the ASFV p54 recombinant protein was successfully expressed by plasmid construction, prokaryotic expression and purification. Monoclonal antibodies (mAbs) were obtained using hybridoma cell technology. Through pair analysis, mAb-6E5 and mAb-4D7 were selected for p54 detection, both exhibiting high affinity and no cross-reactivity with other ASFV proteins. Based on the sandwich colloidal gold immunochromatographic assay principle, a p54 test strip was constructed using 6E5 as the capture antibody and 2D7 as the detection antibody, with a limit of detection of 1.51 ng/mL. The intra- and inter-assay recoveries ranged from 87.8% to 110.6%, with variable coefficient of less than 11.1%. Positive serum samples further confirmed the accuracy of the assay. This developed test strip has the potential to serve as an effective tool for ASFV detection and could play a crucial role in the prevention and management of African Swine Fever (ASF) outbreaks.