Flow cytometry-based multiplexing antibody detection for diagnosis of African swine fever virus

African swine fever (ASF) is an infectious disease that has a mortality rate of nearly 100% in domestic pigs. To date, no vaccine or effective treatment for ASF is available, necessitating the development of an accurate and sensitive diagnostic method to monitor ASF virus (ASFV) antibodies for prevention and control. Herein, a reliable and sensitive suspension microarray technology-based multiplexing method was developed for ASFV antibody detection using recombinant CD2v, p30, p54, and p22 antigen protein coated size-encoded microbeads as probes to capture the target antibody. Compared to commercial ELISA kits, the newly developed method showed a 16-fold improvement in detection sensitivity. Differential diagnosis of CD2v-unpressed low-virulence mutant (genotype II) and wild-type ASFV (genotype II) was readily achieved by fluorescence signal analysis of the CD2v-coated probe in the microbead mixture solution. In addition, the real serum assay revealed a 97% consistency rate between the novel method and commercial ELISA kits, demonstrating excellent potential for ASF epidemic surveillance and control.