Biosensor for Screening Specific Interactions with AhR

用于筛选与 AhR 特异性相互作用的生物传感器

• 批准号: 7066606
• 负责人: Judith L. Erb
• 金额: $ 23.13万
• 依托单位: IA, INC.
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7066606
• 关键词: aromatic hydrocarbon receptor; bioengineering /biomedical engineering; biomedical equipment development; biosensor device; chemical kinetics; dioxins; endocrinology; estrogen receptors; fiber optics; flavonoids; hormone analog; hormone inhibitor; intermolecular interaction; molecular chaperones; toxicant screening

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.

描述（由申请人提供）：在第一阶段工作期间，IA成功地将倏逝传感技术应用于芳烃受体（AhR）介导过程的研究，并区分有益和有害的AhR配体。当使用生物传感器评估 AhR 与配体、辅助蛋白和二恶英反应元件 (DRE) 的相互作用时，每种化合物都会出现独特的传感图。在第二阶段的工作中，将为研究以及环境和食品测试市场开发该应用程序。这将通过以下具体目标来实现： (1) 优化和标准化试剂和方案； (2) 明确文书的详细性质； (3) 建立定量并可靠地区分受体激动剂、拮抗剂和部分激动剂的数据解释方法； (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) 对30-50种化合物验证系统和方法； (7) 获得监管部门批准使用 AhR 生物传感器进行二恶英筛查。研究这些机制的现有方法既麻烦又耗时，对于基于细胞的方法来说，细胞代谢造成的化合物降解和细胞培养物的不稳定性会显着影响结果。在第二阶段，生物传感器仪器和试剂将被优化和原型化，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机制的影响，并根据基于机制的传感器评估来预测化合物的影响。预计传感图的参数化将为区分AhR激动剂、拮抗剂、混合激动剂/拮抗剂和不与AhR相互作用的化合物提供标准。由此产生的生物传感器、试剂和方案将通过使用它们对 30-50 种盲编码化合物进行分类来进行验证。这项工作最初将在 1A, Inc. 完成。该提案包括时间和资源的延长，以支持 4 个独立实验室的类似测试。结果将提交给 ICCVAM 验证流程。该生物传感器系统还将进行测试，以鉴定从五大湖流域获得的真实样品中的二恶英类活性。一旦系统经过验证，它将在食品检测领域具有特殊的价值。欧盟最近的监管立法要求食品经过认证，不含二恶英污染。这将导致向欧盟出口美国食品的价值 65 亿美元的行业需要一种具有成本效益的、快速的现场筛选测试，就像本项目所提供的那样。