The Tadpole Assay Multuplex Platform for Biodefense Agent Diagnostics

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Diagnostic reagents and assays to detect biodefense pathogens are critical needs for public safety. Two key components for a successful molecular diagnostic assay are a sensor component that binds directly to the targeted organism, or to a product secreted by the organism, and a signal domain that indicates, with great sensitivity, when the sensor has bound the target molecule. Over the past 18 months we have implemented proven technologies: phage-display, covalent protein-DNA linkage, and real-time PCR, along with the results from our antigen discovery research, to begin to create a powerful diagnostic assay to detect the presence of Francisella tularensis (Ft) in biological and environmental samples as well as immune responses directed against Ft. The protein-DNA chimeras central to these assays are called tadpoles, which are capable of achieving a much greater level of sensitivity (~106-fold greater) compared to analogous enzyme-linked immunosorbent assays (ELISAs). Furthermore these diagnostics are able to identify target molecules over a wide dynamic range of concentrations. In this proposal, we will multiplex the tadpole assay to include additional fever and diarrheal agents as outlined in the overall WRCE diagnostic theme plan. Ft, Rift Valley fever virus, and Cryptosporidium parvum will serve as initial, comparative controls for the platform WRCE diagnostic approaches, and the agent list will be expanded in later years according to the WRCE plan. Furthermore, we will seek to integrate elemental technologies from other platforms being developed in the WRCE diagnostic group, such as lateral flow microfluidics, to expand the utility of tadpole diagnostics as they are further multiplexed for the simultaneous detection of numerous agents and are adapted for point-of-care usage in addition to their use in the reference laboratory.