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.

项目概要 自身抗体的检测和定量对于 1 型的诊断和管理越来越重要 可以在检测和治疗方面实现临床相关性能的检测方法和系统。 定量 T1D 相关自身抗体——从关键性能参数的角度来看，例如 灵敏度、特异性、重现性和定量准确性——由于一系列因素，已被证明 为了满足这一需求，我们开创了 RAABET（共振自动抗体能量转移）。 T1D 检测，一种快速、免洗多重检测 T1D 相关自身抗体的均质检测 创新的 RAABET 检测是围绕小体积 (1-10 µL) 患者血浆或血清而设计的。 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 具有长寿命、高量子效率的铽穴状化合物福斯特共振能量转移 (FRET) 供体 第二个池具有小分子荧光团 FRET 受体，波长约为 12-13 nm。 两个自身抗体表位结合区域尖端之间的平均距离以及之间约 3.5 nm 的间距 每个表位结合臂的边缘，双池标记方法，当翻译成均质 测定，产生了一个近乎理想的自身抗体特异性 FRET 信号传导架构，并且易于建模 概率供体-受体配对。 已经完成的 RAABET 开发工作包括优化缀合方案， 开发串扰校正算法，提高分析中每个目标物种的定量准确性 多重反应，并进行初步稳定性研究 RAABET 测定的早期版本。 在最近使用多个临床小组进行的初步验证研究中表现非常好 与样本相比，RAABET 在早期测试中的表现尤其引人注目。 ELISA 虽然是一种普遍成熟且高性能的检测方法，但已经被人们所熟知。 对于 T1D 自身抗体，其他 ELISA 替代方法均表现不佳。 存在缺陷，包括分析灵敏度差和/或要求昂贵且难以 维护设备 T1D 自身抗体是一个持续需要的领域； 满足这个需求。 在这个项目中，我们建议采取必要且创新的 RAABET 探针工程举措。 探针工程工作以及相关的验证活动包含在本目标 1 中 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定：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，用于容纳小体积全血样品 RAABET 无需额外的实验室仪器；并表征 RAABET 测定的 表征目标自身抗体的结合亲和力的性能，这是一个额外的功能级别 该检测本质上支持并可以为 T1D 当前和未来的需求提供关键信息 诊断和管理。