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

基于液体微活塞增强时间分辨福斯特共振能量转移的多平台同质多重自身抗体测定

• 批准号: 10576777
• 负责人: Amy Droitcour
• 金额: $ 29.99万
• 依托单位: WAVE 80 BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-15 至 2024-06-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10576777
• 关键词: Address; Advanced Development; Affinity; Algorithms; Antibody titer measurement; Antigens; Architecture; Area; Autoantibodies; Binding; Biochemical; Biological; Biological Assay; Blood; Blood specimen; Cells; Chemistry; Clinical; Complex; Detection; Development; Diabetes autoantibodies; Diagnosis; Disease; Disease Progression; Energy Transfer; Engineering; Enzyme-Linked Immunosorbent Assay; Epitopes; Equipment; Fluorescence Resonance Energy Transfer; Future; Heart; Immune system; Insulin; Insulin-Dependent Diabetes Mellitus; Kinetics; Label; Laboratories; Liquid substance; Measurement; Measures; Methods; Optical Instrument; Optics; Patients; Performance; Phase; Plasma; Preparation; Proteins; Protocols documentation; Reaction; Recombinants; Reproducibility; Running; Sampling; Serum; Signal Transduction; Solid; Source; Specific qualifier value; Specificity; Specimen; Statistical Models; Surface Plasmon Resonance; System; Technology; Terbium; Testing; Time; Translating; Validation; Variant; Whole Blood; Work; arm; clinically relevant; design; detection limit; fluorophore; improved; innovation; instrument; instrumentation; interest; multiplex detection; operation; photonics; point of care; programs; quantum; rapid test; screening; small molecule; stability testing; tool; validation studies

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.

项目摘要 自身抗体的检测和定量越来越优先考虑1型诊断和管理 糖尿病（T1D）。测定方法和系统可以在检测和 定量与T1D相关的自动抗体 - 从关键性能参数的角度（例如 灵敏度，特异性，可重复性和定量准确性 - 由于因素的星座而证明 难以捉摸。为了满足这一需求，我们已经开创了RAABET（共振自动抗体能量转移） T1D分析，一种用于快速，无洗多路复用检测T1D相关自身抗体的均质测定 从小体积（1-10μL）的患者血浆或血清中。创新的Raabet分析是设计的 标签 - 对于每个自身抗体目标 - 每个相关重组抗原的单独池：一个池 具有长寿命的高量化效率terbium隐立替酸Förster共振能量转移（FRET）供体 还有一个带有小分子荧光团特品格受体的池。考虑到大约12-13 nm 两个自身抗体表位结合区域的尖端之间的平均距离与〜3.5nm间距之间的平均距离 每个表位结合臂的边缘，即双池标记的方法，当翻译成同质的边缘 测定，具有特定于自身抗体特异性的FRET信号的几乎理想的结构，并易于建模 概率供体配对。 已经完成的Raabet开发工作包括优化共轭协议， 开发一种串扰校正算法，可提高每个目标物种的定量准确性 多重反应，并进行初步稳定研究。 Raabet分析的早期版本 在最近进行了多个临床面板的初步验证研究中表现出色 样品。与 ELISA虽然是一个普遍建立良好且表现高的测定方法，但已经看到了时间，并且 再次为T1D自身抗体表现不佳。 T1D自动抗体的ELISA的其他替代品all 缺陷，包括分析敏感性差和/或对昂贵且难以难以置信的要求 维护设备。 T1D自动抗体是持续需要的领域； Raabet是最终可以 满足这种需求。 在这个项目中，我们建议采取重要的和创新的雷亚贝特调查工程计划。 探测工程工作以及相关的验证活动被AIM 1所包含 项目。该项目的目标2涵盖了推进创新的Raabet T1D的其他优先事项 测定最终临床验证和产品发布：进行扩展的稳定性测试计划， 开发一种增强的样品制备方法，以适应低量的全血样品 Raabet无需额外的实验室仪器；并描述赖贝特测定法 表征目标自动抗体的绑定亲和力的性能，这是一个额外的功能水平 该测定本质上支持，可以为T1D中的当前和未来需求提供关键信息 诊断和管理。