RAPPID: a platform of ratiometric bioluminescent sensors for homogeneous immunoassays

Ni, Y.; Rosier, B. J. H. M.; van Aalen, E. A.; Hanckmann, E. T. L.; Biewenga, L.; Makri Pistikou, A.-M.; Timmermans, B.; Vu, C.; Roos, S.; Arts, R.; Li, W.; de Greef, T. F. A.; van Kuppeveld, F. J. M.; Bosch, B.-J.; Merkx, M.

doi:10.1101/2020.10.31.363044

bioRxiv · 10.1101/2020.10.31.363044

RAPPID: a platform of ratiometric bioluminescent sensors for homogeneous immunoassays

Ni, Y.·Rosier, B. J. H. M.·van Aalen, E. A.·Hanckmann, E. T. L.·Biewenga, L.·Makri Pistikou, A.-M.·Timmermans, B.·Vu, C.·Roos, S.·Arts, R.·Li, W.·de Greef, T. F. A.·van Kuppeveld, F. J. M.·Bosch, B.-J.·Merkx, M.

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Abstract

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.

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Ni, Y., Rosier, B. J. H. M., van Aalen, E. A., Hanckmann, E. T. L., Biewenga, L., Makri Pistikou, A.-M., Timmermans, B., Vu, C., Roos, S., Arts, R., Li, W., de Greef, T. F. A., van Kuppeveld, F. J. M., Bosch, B.-J., Merkx, M.. 2020-11-03. RAPPID: a platform of ratiometric bioluminescent sensors for homogeneous immunoassays. https://doi.org/10.1101/2020.10.31.363044

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