Biosensor for Screening Specific Interactions with AhR

描述（由申请人提供）：在I期工作期间，IA成功地将瞬逝传感技术应用于芳烃受体（AhR）介导的过程的研究，并提供了有益和有害AhR配体之间的区分。当生物传感器被用来评估AhR与配体，辅助蛋白质和二恶英响应元件（DRE）的相互作用时，出现了每种类型的化合物的独特传感图。在第二阶段的工作中，将为研究和环境及食品测试市场开发这种应用。这将通过以下具体目标实现：（1）优化和标准化试剂和方案;（2）规定仪器的详细性质;（3）建立数据解释方法，以量化和可靠区分受体激动剂、拮抗剂和部分激动剂;（4）改进生物传感器荧光计仪器;（5）改进生物传感器盒的设计和制造;（6）在30-50种化合物上验证该系统和方法;以及（7）获得使用AhR生物传感器进行二恶英筛选的监管批准。研究这些机制的现有方法繁琐且耗时，并且对于基于细胞的方法，细胞代谢引起的化合物降解和细胞培养物的不稳定性显著影响结果。在第二阶段，生物传感器仪器和试剂将进行优化和原型化，并将用于评估配体对影响内分泌干扰化合物之间在受体-DNA相互作用水平上的串扰的机制的影响，并根据基于机制的传感器评估预测化合物的影响。预计传感图的参数化将提供区分AhR激动剂、拮抗剂、混合激动剂/拮抗剂和不与AhR相互作用的化合物的标准。将通过使用它们对30-50种盲编码化合物进行分类来验证所得生物传感器、试剂和方案。这将首先在1A，Inc.完成。该提案包括时间和资源的延长，将支持在4个独立实验室进行类似的测试。结果将提交给ICCVAM验证过程。该生物传感器系统还将进行测试，以确定从五大湖流域获得的真实样本中的二恶英样活性。一旦该系统得到验证，它将在食品检测竞技场中具有特殊的价值。欧盟最近的监管立法要求食品必须证明不受二恶英污染。这将导致价值65亿美元的美国食品出口到欧盟的行业需要一个具有成本效益的，快速的，现场筛选测试，如该项目将提供。

DESCRIPTION (provided by applicant): During Phase I work IA successfully applied evanescent sensing technology to the study of aryl hydrocarbon receptor (AhR)-mediated processes and provided discrimination between beneficial and harmful AhR ligands. When biosensors were used to assess the interaction of AhR with ligands, accessory proteins, and dioxin response element (DRE) distinctive sensorgrams emerged for each type of compound. Under Phase II work applications of this will be developed for the research and the environmental and food-testing markets. This will be achieved through the Specific Aims: (1) Optimize and standardize reagents and protocols; (2) Specify the detailed nature of the instrument; (3) Establish data interpretation methods that quantify and reliably distinguish between receptor agonists, antagonists and partial agonists; (4) Improve biosensor fluorometer instrument; (5) Improve design and manufacturing of biosensor cartridges; (6) Validate the system and methods on 30-50 compounds; and (7) Obtain regulatory approval for use of the AhR biosensor for dioxin screening. Existing methods for studying these mechanisms are cumbersome and time consuming, and for cell-based methods, compound degradation by cellular metabolism and instability of cell cultures significantly affects results. During Phase II, the biosensor instrument and reagents will be optimized and prototyped and will be used to assess the impact of ligands on the mechanisms affecting cross-talk between endocrine disrupting compounds at the level of receptor-DNA interactions and to predict the effects of compounds based on mechanism-based sensor evaluation. It is anticipated that parameterization of sensorgrams will provide criteria for discrimination between AhR agonists, antagonists, mixed agonist/antagonists and compounds which do not interact with AhR. The resulting biosensor, reagents and protocols will be validated by using them to categorize 30-50 blind coded compounds. This will initially be done at 1A, Inc. The proposal incorporates a time and resources extension which will support similar testing in 4 independent laboratories. Results will be submitted to the ICCVAM validation process. The biosensor system will also be tested for identification of dioxin-like activity in real-world samples obtained from the Great Lakes watershed. Once the system is validated it will have particular value in the food testing arena. Recent regulatory legislation by the EU requires that foods be certified free of dioxin contamination. This will cause the $6.5 billion industry which exports U.S. food to the EU to be in need of a cost effective, rapid, on-site screening test such as will be provided by this project.