Develop of Diagnostic Reagents for Detection of Francisella & Francisella Infecti

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Francisella tularensis, the causative agent of tularemia, is one of the most infectious bacteria known and is a Category A biodefense concern. Diagnostic tools that detect Francisella and other select agents are among the critical needs identified by the NIH/NIAID that are targeted for immediate development and are the focus of this proposal. Two key components of a successful diagnostic are a high-affinity sensor domain that binds directly to the targeted organism, or to a product secreted by the organism, and a signal domain that indicates when the sensor has bound the target molecule. We are using proven technologies: single-chain variable domain antibody fragment- (scFv-) phage-display, tadpole protein-DNA chimeras, real-time PCR and the results from our antigen discovery research to create powerful diagnostics that will detect the presence of Francisella tularensis (Ft) in biological and environmental samples as well as immune responses directed against Ft. In this report, we show that a specific tadpole that recognizes mammalian immunoglobulin can be used with a complex biological sample (serum) to detect an immune response mounted against Ft. In initial assays, a Ft-specific immune response is detected in serum that is diluted up to 10,000-fold. This report also includes progress in using phage-display scFv antibody libraries for the development of sensors for Ft-specific targets. In other systems these reagents have been shown to be up to a million-fold more sensitive than what is possible using enzyme-linked immunosorbent assays (ELISA). Furthermore, these diagnostics will be able to identify target molecules over a wide dynamic range of concentrations. This strategy can be applied to the development of diagnostic reagents that recognize any given target and could lead to a pipeline that generates sensitive diagnostic tools for many biodefense/infectious disease pathogens. The bacterium that causes tularemia is one of the most infectious agents known and is a serious biodefense threat. Greatly needed are tools that can detect minute quantities of the organism and/or that can detect whether a person has been infected by the organism early in the course of infection. We are using cutting edge strategies to create molecules that can identify tiny amounts of the organism and that provide a sensitive readout as to the presence or absence of the organism using a common assay. The strategies used to create this powerful tool can easily be adapted to create similar reagents for any biodefense or infectious disease concern.