Are Epitopic Sites of 3AB and 3D Nonstructural Proteins Sufficient for Detection of Foot and Mouth Disease?

Abstract

An efficient method for detection of foot and mouth disease (FMD) and, particularly, differentiation of vaccinated from infected animals is the use of nonstructural (NS) proteins as antigens in Enzyme-Linked Immunosorbent Assay (ELISA) Kits. In this study, only epitopic regions of 3AB and 3D NS proteins were used for recombinant protein production, as a cost-effective method instead of peptide synthesis, for application in in-house ELISA diagnostic kits. Specific primers were designed according to the antigenic regions of 3AB (C-terminus of 3A and the whole 3B) and 3D (N-terminus) proteins, and the polymerase chain reaction (PCR) amplification was performed. Purified amplicons were cloned into pET21a (+) vectors and then transformed into Escherichia coli (BL21). Thereafter, bacteria were induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for expression of antigenic proteins. Antigenic 3AB protein was expressed in soluble form, but 3D protein was extracted from the bacterial lysate. Protein expression was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses. An indirect ELISA was developed for each protein, and the diagnostic sensitivity and specificity were determined. The 3AB-ELISA showed higher sensitivity and specificity than 3D-ELISA (95.24% and 100%, compared with 90.48% and 88.71%, respectively). The epitopic 3AB-ELISA developed here can be used for detection and differentiation of FMD infected from vaccinated animals, but the epitopic 3D-ELISA showed lower efficiency in screening for FMD status.