Rapid and visual dual-color lateral flow immunoassay for african swine fever virus antibody detection

African swine fever (ASF), an acute infectious disease with a high mortality rate, poses considerable challenges to the development of the global swine industry and socio-economic stability. At present, given the lack of commercial vaccines or effective therapeutic methods, the primary strategy for its management relies on rapid diagnosis and culling of infected animals. Thus, there is a pressing need to develop sensitive, simple, and rapid detection methods. In this study, a latex microsphere (LM)-based dual-color lateral flow immunoassay (LFIA) was designed for the detection of African swine fever virus (ASFV) antibody, which was detected within 15 min by the naked eye. Protein 72 (p72) was conjugated with red LM (RLM) and Chicken IgY (CIgY) with blue LM (BLM) to achieve dual-color detection. Moreover, no cross-reactivity was observed between the standard sera of ASFV and other common swine pathogens. The method exhibited a sensitivity of 1:1024, comparable with the commercial ELISA kit. Meanwhile, the evaluation of 159 clinical samples yielded a kappa value of 0.955. Regarding stability, the assay could be stored at 4℃, room temperature (18-25 ℃), and 37 ℃ for 16, 12, and 10 months, respectively. In addition, the intra- and inter-assay variability showed no significant difference, highlighting the excellent repeatability of the method. Overall, these results demonstrate that the dual-color LM-LFIA offers advantages such as low cost, high sensitivity, strong specificity, outstanding stability, and fast detection speed, making it particularly suitable for point-of-care testing (POCT) of ASF. KEY POINTS: • Visually intuitive: Within 15 min, a red C-line indicates a positive ASFV antibody. • Extremely high accuracy: Compared with commercial ASFV antibody ELISA kit, the Cohen's kappa value of this assay is 0.955. • Comparable sensitivity: The detection limit of this assay is 1:1024, matching the performance of a commercial ASFV ELISA kit.