High throughput ELISA chip for drug toxicity screening

描述(由申请人提供)：药物代谢不良、药物间相互作用和毒性效应导致大量药物失效。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磨损。反应生物公司开发了极低成本的纳升反应微阵列，为高温超导在药物发现、大规模IC50测定和选择性分析方面的市场服务。这些反应比目前药物发现中使用的孔板形式小一万倍。微阵列允许在1000‘S裂解产物中测量数百种抗原，这些裂解产物是从用化合物处理的细胞中获得的。这一阶段的具体目标是：目标1：裂解和印刷。设计并实施微阵列表面化学以优化抗体包被的均匀性和稳定性。开发强大的高通量细胞裂解方案，以有效地释放和回收抗原。将为每个微阵列打印的多达6600份裂解产物开发裂解和打印方案。目标2：检测。对每个感兴趣的抗原进行优化和校准，使检测灵敏度降至10pg/ml水平。将测试三种类型的检测，包括：(1)直接荧光二次抗体检测，(2)沉淀酶产物检测，(3)可溶性产酶底物检测。本研究的目的是通过这三种检测方法来检测HepG2裂解产物中的HSP70。目标3：五抗原板。使用目标1中开发的裂解方法和目标2中的检测方法建立、校准和验证5个毒性生物标志物。目标4：药物-药物相互作用。筛选几个测试化合物与HepG2中的药物文库的相互作用，并测量在Aim 3中开发的五个生物标记物。我们寻求创建一个数据库，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一个广泛的蛋白质毒性生物标记物小组和一个完整的通用药库。

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.