Multi-Platform Homogeneous Multiplexed Autoantibody Assay Based on Liquid Micropiston-Enhanced Time-Resolved Forster Resonance Energy Transfer

项目摘要 自身抗体的检测和定量在1型的诊断和治疗中越来越受到重视 糖尿病(T1D)。能够实现临床相关性能的检测方法和系统 量化与T1D相关的自身抗体-从关键性能参数的角度，如 敏感性、特异性、重复性和定量准确性--由于一系列因素，已经证明 难以捉摸。为了满足这一需求，我们开创了RAABET(共振自身抗体能量转移) T1D法，一种快速、免清洗、多重检测T1D相关自身抗体的均相检测方法 从小容量(1-10μL)患者血浆或血清中提取。创新的RAABET检测是围绕以下方面设计的 标记-对于每个自身抗体靶标-每个相关重组抗原的两个单独池：一个池 与长寿高量子效率Tb的Förster共振能量转移(FRET)供体 第二个池具有小分子荧光团FRET受体。给出大约12-13纳米 两个自身抗体表位结合区尖端的平均距离和~3.5 nm的间距 每个表位结合臂的边缘，双池标记方法，当翻译成均相时 实验，为自身抗体特异性FRET信号产生了一个近乎理想的体系结构，易于建模 概率供体-受体配对。 已经完成的RAABET开发工作包括优化共轭方案， 开发串扰校正算法，以提高每个目标物种的定量精度 多重反应，并进行初步的稳定性研究。RAABET试验的早期版本 在最近进行的初步验证研究中表现良好，使用了多个临床小组 样本。RAABET在这一早期测试中的性能与 虽然ELISA法是一种普遍成熟和高效的检测方法，但它已经被发现 再次对T1D自身抗体表现不佳。T1D自身抗体酶联免疫吸附试验的其他替代方法 有不足之处，包括分析灵敏度差和/或要求昂贵和难以- 维护设备。T1D自身抗体是一个长期需要的领域；RAABET是最终可以 满足这一需求。 在这个项目中，我们建议开展基本的和创新的RAABET探测器工程倡议。 探测工程工作以及相关的验证活动包含在本文件的目标1中 项目。该项目的目标2包括推进创新RAABET T1D的其他优先事项 最终临床验证和产品发布的测试：进行扩展的稳定性测试计划， 一种改进的适应低容量全血标本的样品制备方法 RAABET不需要额外的实验室仪器；并表征RAABET检测的 在表征目标自身抗体的结合亲和力方面的性能，这是一种额外的功能级别， 该分析内在地支持并能够为T1D的当前和未来需求提供关键信息 诊断和管理。

Project Summary Detection and quantitation of autoantibodies is increasingly prioritized for diagnosis and management of type 1 diabetes (T1D). Assay methods and systems that can achieve clinically relevant performance in detecting and quantitating T1D-relevant autoantibodies—from the standpoint of key performance parameters such as sensitivity, specificity, reproducibility, and quantitative accuracy—have, due to a constellation of factors, proven elusive. To address this need, we have pioneered the RAABET (Resonance AutoAntiBody Energy Transfer) T1D assay, a homogeneous assay for rapid, no-wash multiplexed detection of T1D-associated autoantibodies from small volumes (1-10 μL) of patient plasma or serum. The innovative RAABET assay is designed around labeling—for each autoantibody target—two separate pools of each associated recombinant antigen: one pool with a long-lived high-quantum-efficiency terbium cryptate Förster Resonance Energy Transfer (FRET) donor and a second pool with a small-molecule fluorophore FRET acceptor. Given the approximately 12-13 nm mean distance between tips of the two autoantibody epitope binding regions and the ~3.5nm spacing between the edges of each epitope binding arm, the dual-pool-labeling approach, when translated to a homogeneous assay, yields a nearly ideal architecture for autoantibody-specific FRET signaling, with readily modeled probabilistic donor-acceptor pairing. RAABET development work that has already been completed has included optimizing conjugation protocols, developing a crosstalk correction algorithm that improves quantitative accuracy for each target species in the multiplex reaction, and conducting preliminary stability studies. The early version of the RAABET assay performed very well in recently conducted preliminary validation studies using multiple panels of clinical samples. The performance of RAABET in this early testing is particularly compelling when compared to ELISA, which, while a generally well-established and high-performing assay method, has been seen time and again to perform poorly for T1D autoantibodies. The other alternatives to ELISA for T1D autoantibodies all have deficiencies, including poor analytical sensitivity and/or requirements for expensive and difficult-to- maintain equipment. T1D autoantibodies is a persistent area of need; RAABET is the assay that can finally meet this need. In this project, we propose to undertake essential—and innovative—RAABET probe engineering initiatives. The probe engineering work, along with associated verification activities, is encompassed by Aim 1 of this project. Aim 2 of this project encompasses additional priorities in advancing the innovative RAABET T1D assay toward final clinical validation and product launch: conducting an expanded stability testing program, developing an enhanced sample preparation method for accommodating low-volume whole blood samples in RAABET without need for additional laboratory instrumentation; and characterizing the RAABET assay’s performance in characterizing binding affinities of target autoantibodies, an additional level of functionality that the assay inherently supports and that can provide critical information for current and future needs in T1D diagnosis and management.