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. Many current immunoassays require multiple washing, incubation and optimization steps. Here the authors present Ratiometric Plug-and-Play Immunodiagnostics (RAPPID), a generic assay platform that uses ratiometric bioluminescent detection to allow sandwich immunoassays to be performed directly in solution.
【저자키워드】 ELISA, Assay systems, Biochemical assays, Fluorescent proteins, Bioanalytical chemistry, 【초록키워드】 antibodies, Biomarkers, translation, antibody, media, ELISA, Spike protein, immunoassays, Protein, immunoassay, point-of-care, anti-SARS-CoV-2 antibodies, SARS-CoV-2 spike protein, therapeutic, Incubation, optimization, platform, blood plasma, therapeutic antibodies, clinical laboratories, solution, complex, luciferase, quantitative measurement, Nanoluc luciferase, robust, performed, provide, homogeneous, bioluminescent, calibrator, 【제목키워드】 immunoassay, platform, homogeneous, bioluminescent,