RAPPID: a platform of ratiometric bioluminescent sensors for homogeneous immunoassays

Ni, Yan; Rosier, Bas J.H.M.; van Aalen, Eva A.; Hanckmann, Eva T.L.; Biewenga, Lieuwe; Pistikou, Anna-Maria Makri; Timmermans, Bart; Vu, Chris; Roos, Sophie; Arts, Remco; Li, Wentao; de Greef, Tom F.A.; van Kuppeveld, Frank J. M.; Bosch, Berend-Jan; Merkx, Maarten

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into easy-to-use 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 that can be detected using a basic digital camera. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. We also introduce the use of a calibrator luciferase that provides a robust ratiometric signal, allowing 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. RAPPID combines ratiometric bioluminescent detection with antibody-based target recognition into an easy-to-implement standardized workflow, and therefore represents an attractive, fast, and low-cost alternative to traditional immunoassays, both in an academic setting and in clinical laboratories for point-of-care applications.