We have developed a homogeneous time-resolved Förster Resonance Energy Transfer (TR-FRET) assay for rapid, no-wash multiplexed detection of T1D-associated autoantibodies from small volumes (5-10 μL) of patient plasma or serum. In our assay, separate pools of recombinant antigen are labelled with either a long-lived high-quantum-efficiency terbium cryptate FRET donor or a small-molecule flurophore FRET acceptor, generating potent FRET pairs. With the mean distance between the tips of the two epitope binding regions approximately 12-13 nm and the distance between the edges of each epitope binding arm on the order of 3.5 nm, the pairs are brought into signaling proximity when the twin binding arms of immunoglobulin bind a donor and an acceptor. We used terbium’s tightly banded multi-peak luminescence with large stokes shift and long millisecond-scale lifetime under 350 nm excitation to engineer the assay for detection of four T1D targets at clinically relevant concentrations—GAD 65, IA-2, ZnT8 and insulin—along with a fifth pair as an internal control. We report on the assay’s impressive performance with clinical samples, including the development and implementation of a high-performance crosstalk correction algorithm. The assay described here functions identically in both commercial plate readers and in point-of-care systems, a key feature for highly consistent test results.
Disclosure

A.A. Metz: Employee; Self; Wave 80 Biosciences, Inc. A.M. Arsham: Consultant; Self; Wave 80 Biosciences. D.J. Laser: Board Member; Self; Wave 80 Biosciences. A.D. Droitcour: Employee; Self; Wave 80 Biosciences.

Funding

Type 1 Diabetes Foundation

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