High throughput ELISA chip for drug toxicity screening

描述（由申报人提供）：药物代谢不良、药物-药物相互作用和毒性作用导致许多药物失败。基于化学结构预测药物毒性的高通量筛选（HTS）技术有可能减少管道损耗。反应生物公司开发了极低成本的纳米升反应微阵列，以服务于HTS在药物发现、大规模IC50测定和选择性分析方面的市场。这些反应比目前用于药物发现的孔板形式小10000倍。微阵列允许在1000个化合物处理的细胞裂解物中测量数百种抗原。这个阶段的具体目标是：目标1：裂解和打印。设计和实现微阵列表面化学，以优化抗体涂层的均匀性和稳定性。开发稳健的高通量细胞裂解方案，以实现高效的抗原释放和恢复。裂解和打印协议将为每个微阵列打印多达6600个裂解物而开发。目标2：检测。优化和校准检测灵敏度低至10 pg/ml的每个抗原感兴趣的水平。将测试三种类型的检测，包括：(1)直接荧光二抗检测，(2)沉淀酶产物，(3)可溶性荧光酶底物。本实验将通过三种检测方法检测HepG2裂解物中的HSP70。目标3：五抗原小组。使用Aim 1中开发的裂解方法和Aim 2的检测方法建立、校准和验证5种毒性生物标志物。目标4：药物-药物相互作用。筛选几种测试化合物与HepG2中药物文库的相互作用，并测量Aim 3中开发的五种生物标志物。我们试图建立一个数据库，其中包括候选药物的化学信息和Aim 4产生的蛋白质表达信息，以建立结构-毒性关系（STR）。在第二阶段，RBC将把这项技术扩展到广泛的蛋白质毒性生物标志物和完整的常见药物库。

DESCRIPTION (provided by applicant): Adverse drug metabolism, drug-drug interactions, and toxicity effects cause numerous drug failures. High throughput screening (HTS) technologies to predict drug toxicity based on chemical structures have the potential to reduce pipeline attrition. Reaction Biology Corporation has developed extremely low cost nanoliter reaction microarrays to serve markets for HTS in drug discovery, large scale IC50 determinations, and selectivity profiling. These reactions are 10,000-fold smaller than well plate formats currently used in drug discovery. Microarrays allow hundreds antigens to be measured in 1000's of lysates obtained from cells treated with compounds. Specific aims in this Phase I are: Aim 1: Lysis and Printing. Design and implement microarray surface chemistry for optimized antibody-coating uniformity and stability. Develop robust high throughput cellular lysis protocols for efficient antigen release and recovery. Lysis and printing protocols will be developed for up to 6600 lysates printed per microarray. Aim 2: Detection. Optimize and calibrate detection sensitivity down to the 10 pg/ml level for each antigen of interest. Three types of detection will be tested, including: (1) direct fluorescent secondary antibody detection, (2) precipitated enzyme products, and (3) soluble fluorogenic enzyme substrates. This aim will measure HSP70 in HepG2 lysates by the three detection methods. Aim 3: Five-antigen Panel. Establish, calibrate, and validate 5 toxicity biomarkers using the lysis methods developed in Aim 1 and detection methods of Aim 2. Aim 4: Drug-drug interaction. Screen several test compounds for interactions with a pharmaceutical library in HepG2 and measure the five biomarkers developed in Aim 3. We seek to create a database that includes the chemical information of the drug candidates and the protein expression information generated from Aim 4 to establish structure-toxicity relationships (STR). In Phase II, RBC will expand this technology to an extensive panel of protein toxicity biomarkers and a full common drug library.