African swine fever (ASF) is a lethal, hemorrhagic, and highly contagious infectious disease caused by the African swine fever virus (ASFV). At present, ASFV has evolved into multiple variant strains with different levels of virulence in China, complicating the epidemiological landscape. In the absence of effective commercial vaccines, the development of rapid and accurate diagnostic methods has become a critical priority for epidemic prevention and control. In this study, monoclonal antibodies (mAbs) were generated by immunizing mice with inactivated ASFV. Nine newly identified mAbs targeting the p72 protein were screened to establish a colloidal gold immunochromatographic strip for ASFV detection combined with 11 previously characterized anti-p72 mAbs developed in our laboratory. Cross-pairing analysis was subsequently performed to identify the optimal antibody combination. Based on this evaluation, ASFV-3 and P72-6 were selected as the gold-conjugated antibody and capture antibody, respectively. The assay demonstrated the capacity to effectively detect diverse ASFV genotypes, with a limit of detection (LOD) of 102.50 TCID50 per reaction against genotype Ⅰ and Ⅱ ASFVs. No cross-reactivity was observed with five common swine pathogens including PCV, PRRSV, PRV, PEDV, and TGEV. The concordance rate with the results from commercially available test strips was 93.33%. In conclusion, a rapid, sensitive, and highly specific colloidal gold immunochromatographic strip targeting the ASFV p72 protein was successfully developed. This method is simple to operate, does not require specialized equipment, and is suitable for on-site detection, providing a valuable tool for field surveillance and early diagnosis of ASF.

Keywords: African Swine Fever Virus; colloidal gold immunochromatography strip; detection; p72 protein.