Development of a dot immunoblot method for differentiation of animals infected with foot-and-mouth disease virus from vaccinated animals using non-structural proteins expressed prokaryotically

Five non-structural proteins (NSPs) of foot-and-mouth disease virus (FMDV) were expressed in E. coli to develop a dot immunoblot (dot blot) assay for the differentiation of FMDV infected animals from vaccinated animals (DIVA). The five NSPs were 3A (24 kDa), 3B (15 kDa), major B-cell epitope regions of 2C (23 kDa), partial 3D (44 kDa) and 3ABC (59 kDa). The criteria for the dot blot were determined and are described as follows: a test sample is considered positive if four or more NSPs demonstrate staining densities equal to or higher than those of their appropriate controls; a sample is considered negative if two or more antigens demonstrate densities below their negative control. A specificity of 100% was observed based on testing of sera from clinical healthy animals with or without vaccination; the sensitivity of the dot blot was 96.1% and 65.8% for testing of samples from infected cattle and swine, respectively, at an early stage of the infection. Meanwhile, high rates of concordance were observed between the dot blot and the PrioCHECK® FMDV-NS test. The dot blot has the potential to act as a confirmatory method for DIVA by 3ABC-ELISA.