Autoantibody- and microRNA-based Next-Gen Multiplex Test for Type 1 Diabetes

 描述(申请人提供)：糖尿病是美国发病率和死亡率的主要原因。鉴于糖尿病在全球范围内的流行继续增长，显然需要改进对疾病及其并发症的管理。早期诊断和干预可明显改善远期预后。抗胰岛自身抗体是1型糖尿病(T1D)患者的血清学标志。此外，最近发现循环中的microRNAs(MiRNAs)发生了系统性的改变，表明miRNA特征具有诊断作用。越来越多的证据表明，与单标记物检测相比，包含具有高临床敏感性和特异度的生物标记物组合的多标记物检测策略可能会提高未来诊断和预后的准确性。为了支持识别信息量最大的生物标志物面板的努力，需要可靠的下一代平台技术，允许对小样本中的标志物进行多路检测，并适合于自动化和整合到临床实验室工作流程中。Nesher Technologies，Inc.(NTI)已独家授权一种超灵敏和特异的生物检测技术的知识产权，该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，具有高单井多路复用潜力、最少的样品要求和简化的处理程序(无需分离/洗涤和放大步骤)。它基于交替激光激发(Alex)单分子荧光光谱，目标识别分子被不同颜色的荧光染料(和猝灭剂)标记。NTI最近实现了从2色到4色Alex的扩展，大大扩展了其多路复用能力，并展示了对直接蛋白质和miRNA定量的诊断实用。此外，最近的工作，由教授。Steve Quake和Shimon Weiss展示了i)将基于微流体的样品处理与Alex光谱相结合，称为“单分子光流体”，以及ii)使用多焦点激发/检测几何结构来提高吞吐量。NTI的长期目标是开发快速、高度多元化、超灵敏和特异的、以及全自动的、基于核酸和蛋白质的诊断测试，这些测试需要最小的样本量。在这里，我们建议开发和临床验证下一代测试，显著改善诊断、预后和治疗指导特性，实施一组自身抗体和miRNA生物标记物，并克服当前T1D测试的局限性。我们的具体目标是：1.为T1D-2的基于自身抗体和miRNA的下一代测试开发初始试剂。使用添加的样本进行分离和多重生物标记物的检测和量化。3.对200个存档的临床样本进行基于Alex的分析，并对ELISA和qPCR方法进行交叉验证。SBIR第二阶段将建议扩大检测范围，以包括更多的标记物，小型化，并开发一个用户友好的“样本输入-答案”诊断系统，提供显著的成本和患者样本节省。

 DESCRIPTION (provided by applicant): Diabetes is a major cause of morbidity and mortality in the U.S. Given that the diabetes epidemic continues to grow worldwide, there is a clear need for improvements in the management of the disease and its complications. Early diagnosis and intervention can significantly improve long-term prognosis. Autoantibodies against islet antigens are a serologic hallmark of patients with Type 1 Diabetes (T1D). In addition, circulating microRNAs (miRNAs) have been shown recently to be systematically altered, indicative of miRNA signatures with diagnostic utility. Growing evidence suggests that a multi-marker strategy, containing a combination of biomarkers with high clinical sensitivity and specificity, may enhance diagnostic and prognostic accuracy in the future compared to single marker tests. To support efforts of identifying the most informative biomarker panels, reliable next- generation platform technologies are needed that permit multiplexed detection of markers in small samples and are suitable for automation and integration into the clinical laboratory work flow. Nesher Technologies, Inc. (NTI) has exclusively licensed the intellectual property for an ultrasensitive and - specific biodetection technology, developed at the UCLA Single Molecule Biophysics Lab (headed by Prof. Shimon Weiss), with high single-well multiplexing potential, minimal sample requirements, and simplified handling procedures (no separation/washing and amplification steps). It is based on alternating laser excitation (ALEX) single molecule fluorescence spectroscopy, whereby target recognition molecules are tagged with different color fluorescent dyes (and quenchers). NTI recently achieved extension from 2-color to 4-color ALEX, substantially expanding its multiplexing power, and demonstrated diagnostic utility for direct protein as well as miRNA quantification. Furthermore, recent work by Profs. Steve Quake and Shimon Weiss shows i) combination of microfluidics-based sample handling with ALEX spectroscopy, 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, ultrasensitive and -specific, as well as fully automated, nucleic acid- and protein-based diagnostic tests that require minimal sample sizes. Here, we propose assay development and clinical validation of a next-generation test with significantly improved diagnostic, prognostic, and treatment- guiding properties, implementing a panel of autoantibody and miRNA biomarkers, and overcoming limitations of current T1D testing. Our Specific Aims are: 1. Initial reagent development for a multiplex autoantibody- and miRNA-based next-generation test for T1D 2. Separate as well as multiplexed biomarker detection and quantification using spiked samples 3. ALEX-based analysis of 200 archived clinical samples and cross-validation to ELISA and qPCR methods SBIR Phase II will propose assay expansion to include more markers, miniaturization, and development of a user-friendly, "sample in - answer out" diagnostic system offering significant cost and patient sample savings.