Multiplex ALEX-based Test for Protease Activity Profiling in Arthritic Joints

描述(申请人提供)：关节炎是一种‘整个关节’的异质性疾病，不同的蛋白水解酶组合在不同的患者亚群中活跃，导致进行性关节退化。前期识别和分层 对于特定抑制剂的适当干预至关重要，其中几种正在临床试验中进行评估。Nesher Technologies，Inc.(NTI)建议使用交替激光激发(Alex)单分子荧光光谱技术，对关节炎关节的微小滑液样本中的蛋白酶活性进行下一代多重测试。这项测试应该被证明是非常有用的，以促进治疗(以及临床试验)使用合适的蛋白酶抑制剂或其组合，根据个别患者的个人资料的关节炎治疗。NTI独家授权了Alex的知识产权，这是一项革命性的定量、超灵敏和特定的生物检测技术，由加州大学洛杉矶分校单分子生物物理实验室(由Shimon Weiss教授领导)开发，具有精致的单井多路复用潜力，最少的样品要求，以及极其简化的处理程序(无需分离/洗涤和放大步骤)。目标识别分子用不同颜色的荧光染料(和猝灭剂)标记。基于共聚焦显微镜，它可以超灵敏地检测溶液中的生物分子，同时区分多个目标，并通过分子计数进行直接、无扩增的定量。NTI最近实现了从2色(2c)到4色(4c)Alex的扩展，大大扩展了其多路复用能力，并展示了对蛋白质和核酸检测的诊断实用。此外，我们的顾问教授最近的工作。Steve Quake和Shimon Weiss展示了i)基于微流体的样品处理与Alex光谱的结合，这是一种称为“单分子光流体”的分析小型化的新突破性方法，以及ii)使用多焦点激发/检测几何结构提高了吞吐量。NTI的长期目标是开发快速、高度多样化(每个样本有100个分析物的能力)、超灵敏和特异的、定量的、成本效益高的和需要最少的全自动诊断测试 样本大小。对于这项SBIR第一阶段拨款申请，我们建议对MMP-13和ADAMTS-4的蛋白酶活性进行多路检测和量化，作为原则证明，在第二阶段将扩展到最多10种酶的小组。我们的具体目标是：1.基质金属蛋白酶-13和ADAMTS-4底物多肽的合成和荧光团/猝灭剂的偶联；2.分离和多重的蛋白酶活性检测和定量；3.基于ALEX的42份临床滑液样本的分析(探针研究设计和实施)。第二阶段将致力于分析扩展、微型化和仪器原型开发。后续的技术商业化将瞄准制药、诊断和基础研究市场。

DESCRIPTION (provided by applicant): Arthritis is a heterogeneous disease of the 'whole joint', whereby different combinations of proteolytic enzymes are active in different patient subpopulations leading to progressive joint degeneration. Upfront identification and stratification is critical for appropriate intervention with specific inhibitors, several of which are being evaluated in clinical trials. Nesher Technologies, Inc. (NTI) proposes a next-generation multiplex test for protease activity profiling in minute synovial fluid samples from arthritic joints, using alternating laser excitation (ALEX) single molecule fluorescence spectroscopy. This test should prove highly useful for facilitating treatment (as well as clinical trials) of arthritis with suitale protease inhibitors, or combinations thereof, personalized according to an individual patient's profile. NTI has exclusively licensed the intellectual property for ALEX, a revolutionary quantitative, ultrasensitive and -specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with exquisite single-well multiplexing potential, minimal sample requirements, and extremely simplified handling procedures (no separation/washing and amplification steps). Target recognition molecules are tagged with different color fluorescent dyes (and quenchers). Based on confocal microscopy, it allows ultrasensitive detection of biomolecules in solution, differentiation of numerous targets simultaneously, and direct, amplification-free quantification via molecule counting. NTI recently achieved expansion from 2- color (2c) to 4-color (4c) ALEX, substantially expanding its multiplexing power, and demonstrated diagnostic utility for detection of proteins and nucleic acids. Furthermore, recent work by our consultants Profs. Steve Quake and Shimon Weiss shows i) combination of microfluidics-based sample handling with ALEX spectroscopy, a new breakthrough approach for assay miniaturization termed "single molecule optofluidics", and ii) enhanced throughput using a multifoci excitation/detection geometry. NTI's long-term goal is to develop rapid, highly multiplexed (with a capacity of >100 analytes per sample), ultrasensitive and -specific, quantitative, cost-effective, and fully automated diagnostic tests requiring minimal sample sizes. For this SBIR Phase I grant application we propose multiplexed detection and quantification of protease activities for MMP- 13 and ADAMTS-4 as proof-of-principle, to be expanded in Phase II to a panel of up to 10 enzymes. Our Specific Aims are: 1. MMP-13 and ADAMTS-4 substrate peptide synthesis and fluorophore/quencher conjugation; 2. Separate as well as multiplexed protease activity detection and quantification using spiked samples; 3. ALEX-based analysis of 42 archived clinical synovial fluid samples (PRoBE study design implementation). Phase II will be dedicated to assay expansion, miniaturization, and instrument prototype development. Sub- sequent technology commercialization will target pharmaceuticals, diagnostics, and basic research markets.