Develop of Diagnostic Reagents for Detection of Francisella & Francisella Infecti

图拉氏弗朗西斯杆菌是图拉热症的病原体，是已知的最具传染性的细菌之一，是一种 A类生物防御问题。检测弗朗西塞氏菌和其他特定病原体的诊断工具包括 NIH/NIAID确定的旨在立即发展并成为重点的关键需求 这项提议。成功诊断的两个关键组件是结合高亲和力的传感器结构域 直接到目标有机体，或到由有机体分泌的产物，以及 指示传感器何时与目标分子结合。我们使用的是成熟的技术：单链 可变区抗体片段-(scFv-)噬菌体展示，蝌蚪蛋白-DNA嵌合体，实时荧光定量PCR 我们的抗原发现研究的结果创造了强大的诊断方法，将检测到 土拉方杆菌在生物和环境样品中的存在及其免疫反应 直指堡垒。 在这份报告中，我们展示了一种识别哺乳动物免疫球蛋白的特定蝌蚪可以与 一种复杂的生物样本(血清)，用于检测针对Ft.在最初的化验中， 在稀释到10,000倍的血清中检测到FT特异性免疫反应。该报告还包括 噬菌体展示单链抗体抗体库在Ft特异性传感器研究中的应用进展 目标。在其他系统中，这些试剂的灵敏度已被证明比 使用酶联免疫吸附试验(ELISA法)是可能的。此外，这些诊断将 能够在很大的动态浓度范围内识别目标分子。这个策略可以是 应用于诊断试剂的开发，这种试剂可以识别任何给定的靶点，并可能导致 为许多生物防御/传染病病原体生成敏感诊断工具的管道。 引起图拉热症的细菌是已知的最具感染性的病原体之一，是一种严重的生物防御。 威胁。非常需要能够检测到微量有机体和/或能够检测到 一个人是否在感染过程的早期被这种微生物感染。我们正在使用切割 边缘策略，以创造分子，可以识别微量的有机体，并提供 用普通的化验方法灵敏地读出有机体的存在或不存在。策略 用于创建这种强大的工具可以很容易地改装为创建任何生物防御或类似的试剂 传染病方面的担忧。

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.