A plug-and-play platform of ratiometric bioluminescent sensors for homogeneous immunoassays
Yan Ni, Bas J. H. M. Rosier, Eva A. van Aalen, Eva T. L. Hanckmann, Lieuwe Biewenga, Anna‐Maria Makri Pistikou, Bart Timmermans, Chris Vu, Sophie Roos, Remco Arts, Wentao Li, Tom F. A. de Greef, Marcel M. G. J. van Borren, Frank J. M. van Kuppeveld, Berend‐Jan Bosch, Maarten Merkx
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.