A plug-and-play platform of ratiometric bioluminescent sensors for homogeneous immunoassays

Yan Ni, Bas J. H. M. Rosier, Eva A. Aalen, Eva T. L. Hanckmann, Lieuwe Biewenga, Anna-Maria Makri Pistikou, Bart Timmermans, Chris Vu, Sophie Roos, Remco Arts, Wentao Li, Tom F. A. Greef, Marcel M. G. J. Borren, Frank J. M. Kuppeveld, Berend-Jan Bosch and Maarten Merkx ()
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Yan Ni: Eindhoven University of Technology
Bas J. H. M. Rosier: Eindhoven University of Technology
Eva A. Aalen: Eindhoven University of Technology
Eva T. L. Hanckmann: Eindhoven University of Technology
Lieuwe Biewenga: Eindhoven University of Technology
Anna-Maria Makri Pistikou: Eindhoven University of Technology
Bart Timmermans: Eindhoven University of Technology
Chris Vu: Eindhoven University of Technology
Sophie Roos: Eindhoven University of Technology
Remco Arts: Eindhoven University of Technology
Wentao Li: Utrecht University
Tom F. A. Greef: Eindhoven University of Technology
Marcel M. G. J. Borren: Rijnstate Hospital
Frank J. M. Kuppeveld: Utrecht University
Berend-Jan Bosch: Utrecht University
Maarten Merkx: Eindhoven University of Technology

Nature Communications, 2021, vol. 12, issue 1, 1-12

Abstract: Abstract Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a mix-and-measure homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. Introduction of a calibrator luciferase provides a robust ratiometric signal that allows direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. With its easy-to-implement standardized workflow, RAPPID provides an attractive, fast, and low-cost alternative to traditional immunoassays, in an academic setting, in clinical laboratories, and for point-of-care applications.

Date: 2021
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DOI: 10.1038/s41467-021-24874-3

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