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: 6786886
• 负责人: Judith L. Erb
• 金额: $ 43.81万
• 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/6786886
• 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在研究芳基烃受体（AHR）介导的过程的研究中成功地应用了Evanescent Sensing技术，并提供了有益和有害AHR配体的歧视。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，每种类型的化合物都出现了独特的传感器。根据第二阶段的工作应用，将为研究以及环境和食品测试市场开发。这将通过特定目的来实现：（1）优化和标准化试剂和协议； （2）指定仪器的详细性质； （3）建立数据解释方法，以量化和可靠地区分受体激动剂，拮抗剂和部分激动剂； （4）改善生物传感器荧光计仪器； （5）改善生物传感器墨盒的设计和制造； （6）在30-50种化合物上验证系统和方法； （7）获得使用AHR生物传感器进行二恶英筛选的法规批准。研究这些机制的现有方法是繁琐且耗时的，对于基于细胞的方法，细胞代谢和细胞培养的不稳定性的复合降解会显着影响结果。在II期期间，将对生物传感器仪器和试剂进行优化和原型，并将用于评估配体对受体-DNA相互作用水平上内分泌干扰化合物之间影响交叉言论的机制的影响，并预测基于机制传感器评估的化合物的影响。预计传感器的参数化将为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歧视的标准。所得的生物传感器，试剂和协议将通过使用30-50个盲型化合物进行验证。最初将在1A，Inc。进行。该提案包含时间和资源扩展，该时间和资源将支持4个独立实验室中的类似测试。结果将提交给ICCVAM验证过程。生物传感器系统还将进行测试，以鉴定从大湖区获得的现实世界样品中的二恶英样活性。一旦系统验证，它将在食品测试领域具有特殊的价值。欧盟的最新监管立法要求将食物获得没有二恶英污染的认证。这将导致65亿美元的行业将美国食品出口到欧盟需要经济有效，快速，现场筛查测试，例如该项目将提供。