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.
Highlights
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
The broad scope and excellent analytical performance of RAPPID are demonstrated by developing assays for a range of clinically relevant biomarkers, including cardiac troponin I, C-reactive protein (CRP), three anti-drug-antibodies (ADAs), and two therapeutic antibodies, displaying robust increases in emission ratio of up to 36-fold
To establish a universally applicable sensing platform, we employed protein G-based photoconjugation for the synthesis of sensor components. This enables direct conjugation to most commercially available primary antibodies, obviating the use of labeled secondary antibodies which increases complexity and can decrease the efficiency of sandwich complex formation[24]. Both the 18-kDa large BiT (LB) and the 1.3-kDa small BiT (SB) fragment of split NanoLuc were genetically fused via a semi-rigid peptide linker to a protein G adapter domain (Gx), carrying the photocrosslinkable nonnatural amino acid p-benzoylphenylalanine (Gx-LB and Gx-SB, respectively, see Fig. 1b and Supplementary Fig. 5)
Summary
To establish a universally applicable sensing platform, we employed protein G-based photoconjugation for the synthesis of sensor components This enables direct conjugation to most commercially available primary antibodies, obviating the use of labeled secondary antibodies which increases complexity and can decrease the efficiency of sandwich complex formation[24]. To establish proof of principle and characterize the performance of the RAPPID sensor platform, we first developed a sensor targeting cardiac troponin I (cTnI), an important protein biomarker for a myocardial injury that requires highly sensitive detection at picomolar concentrations (Fig. 2a)[30] To this end, Gx-LB and Gx-SB were photoconjugated to a pair of commercially available anti-cTnI antibodies with distinct binding epitopes (19C7 (mIgG2b) and 4C2 (mIgG2a), respectively, Fig. 1b). These observations indicate that the thermodynamic a RAPPID for b
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