Assessment of the diagnostic potential of immuno-RCA in 96-well ELISA plates for foot-and-mouth disease virus

The need for fast and very early detection of foot-and-mouth disease virus (FMDV) infection has yielded different types of diagnostic tools over the past decades: whereas very sensitive techniques such as virus isolation (VI) and more recently also real-time RT-PCR can provide evidence for the presence of low virus quantities, VI requires additional confirmation of the nature of the virus strain and both techniques (currently) lack the ability for direct serotyping. The latter usually depends on ELISA, which is a far less sensitive method and may require virus culturing. This paper elaborates on experimental efforts towards the development of an ‘immuno-rolling circle amplification (RCA)’ assay in 96-well plates, the aim being to increase the sensitivity of immunological FMDV detection and serotyping by means of RCA. The study attempts to explain the encountered hurdles and the complexity of the different setups tested. Conclusively, immuno-RCA in 96-well plates as a reliable diagnostic assay for FMDV seems very difficult to achieve.