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

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.