RAPPID: a platform of ratiometric bioluminescent sensors for homogeneous immunoassays

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.

Toward a Point-of-Need Bioluminescence-Based Immunoassay Utilizing a Complete Shelf-Stable Reagent

“…In comparison to fluorescence technologies, the low background signal for bioluminescence reporters results in high sensitivity and a broad dynamic range, which is advantageous for the development of homogenous assay formats for point-of-need applications. , NanoLuc (Nluc), a small (19 kDa), 10-strand β-barrel luciferase − that utilizes the furimazine (Fz) substrate to produce bright and sustained luminescence, is the basis for a binary complementation-based molecular interaction reporter known as NanoBiT. , The NanoBiT system consists of an 18 kDa polypeptide (LgBiT; Nluc β-strands 1–9) that was engineered for stability and a small 11mer peptide (β-strand 10 of Nluc), which can be used as complementation reporter moieties for analyzing protein interactions or for the sensitive detection of tagged proteins. Both full-length Nluc as well as NanoBiT have been applied as strategies for a variety of interesting analyte detection systems with potential relevance to point-of-need assays. ,− …”

A Genetically Encoded Bioluminescent System for Fast and Highly Sensitive Detection of Antibodies with a Bright Green Fluorescent Protein

“…SARS-CoV-2 has infected over 240 million people and caused more than 4.8 million deaths over the world until October 17, 2021 . Currently, most diagnostic tests for SARS-Cov-2 rely on RT-PCR amplification of viral RNA from patient swabs and immuno-detection of virus-specific antibodies or structural proteins in blood, , the latter of which includes Nluc-based nonratiometric − and ratiometric bioluminescent approaches . However, the ratiometric system RAPPID, which is based on SARS-CoV-2 spike protein-induced complementation of split Nluc fragments fused to protein G, requires four affinity chromatography purifications and covalent photoconjugation of Nluc fragments to an antibody sandwich pair via protein G. Therefore, we explored the possibility of using the highly sensitive Clover4-LUMABS to monitor specific antibodies against SARS-Cov-2 in serum.…”

Rethinking Our COVID-19 Strategy

“…The Need for Improved POC Testing There have been calls to rethink our testing strategy, as shown by a thought-provoking paper by Mina et al [20]. POC [45], while a saliva-based test that can yield results within seconds is also being commercialized. (www.blinkscience.com) It is beyond this paper's scope to go into testing technologies in more detail, but it is clear that several options are becoming available that make large-scale and rapid testing of the population feasible.…”