Simultaneous detection of antibodies to foot-and-mouth disease non-structural proteins 3ABC, 3D, 3A and 3B by a multiplexed Luminex assay to differentiate infected from vaccinated cattle.

For the first time, a multiplex bead immunoassay was used to test simultaneously, with a single sample, the immune response to foot-and-mouth disease non-structural proteins 3ABC, 3A, 3B and 3D from experimentally infected and vaccinated cattle. We cloned and expressed these non-structural proteins (NSPs) as recombinant antigens. The purified proteins were coupled to microspheres labeled with anti-His monoclonal antibody with different proportions of red and orange fluorescent dyes and reacted against serum specimens. Antibody reacting against different NSPs, and thus, the different colored beads, was detected by use of the Luminex system. This multiplex bead immunoassay can detect the immune response to NSPs in cattle as early as 7 days post-infection. In general antibodies to the protein 3D appeared early after infection and anti-3ABC antibodies were detected at higher levels than the other NSPs. A clear differentiation was established between infected and vaccinated or uninfected cattle. The multiplex bead immunoassay was compared to individual indirect enzyme-linked immunosorbent assays (iELISAs) for the same NSP's responses. Results indicated that this new assay had a high positive correlation with those generated by iELISA. The Luminex-based technology promises to be a sensitive and efficient method that permits multiplexed NSP antibody detection from a single sample and would therefore provide both a time and cost saving to the laboratory.