Design of multiplexing lateral flow immunoassay for detection and typing of foot-and-mouth disease virus using pan-reactive and serotype-specific monoclonal antibodies: Evidence of a new hook effect

The foot-and-mouth disease (FMD) is the most important transboundary viral disease of livestock in the international context, because of its extreme contagiousness, widespread diffusion, and severe impact on animal trade and animal productions. The rapid and on-field detection of the virus responsible for the FMD represents an urgent demand to efficiently control the diffusion of the infection, especially in low resource setting where the FMD is endemic. Colorimetric lateral flow immunoassay (LFIA) is largely used for the development of rapid tests, due to the extreme simplicity, cost-effectiveness, and on-field operation. In this work, two multiplex LFIA devices were designed for the diagnosis of FMD and the simultaneous identification of major circulating serotypes of the FMD virus. The LFIAs relied on the sandwich-type immunoassay and combined a set of well-characterised monoclonal antibodies (mAb) pairs. One LFIA aimed at detecting and identifying O, A and Asia-1 serotypes, the second device enabled the detection and differentiation of the SAT 1 and SAT 2 serotypes. Both devices also incorporated a broad-specific test line reporting on infection from FMDV, regardless the strain and the serotype involved. Accordingly, five and four reactive zones were arranged in the two devices to achieve a total of six simultaneous analyses. The development of the two multiplex systems highlighted for the first time the relevance of the mAb positioning along the LFIA strip in connection with the use of the same or different mAb as capture and detector ligands. In fact, the excess of detector mAb typically employed for increasing the sensitivity of sandwich immunoassay induced a new type of hook effect when combined with the same ligand used as the capture. This effect strongly impacted assay sensitivity, which could be improved by an intelligent alignment of the mAb pairs along the LFIA strip. The analytical and diagnostic performances of the two LFIAs were studied by testing reference FMDV strains grown in cell cultures and some representative field samples (epithelium homogenates). Almost equivalent sensitivity and specificity to those of a reference Ag-ELISA kit were shown, except for the serotype SAT 2. These simple devices are suitable in endemic regions for in-field diagnosis of FMD accompanied by virus serotyping and, moreover, could be deployed and used for rapid confirmation of secondary outbreaks after FMD incursions in free-areas, thus contributing to promptly implement control measures.