Turning Antibodies into Ratiometric Bioluminescent Sensors for Competition-Based Homogeneous Immunoassays

Abstract

Here we present LUCOS (Luminescent Competition Sensor), a modular and broadly applicable bioluminescent diagnostic platform enabling the detection of both small molecules and protein biomarkers. The construction of LUCOS sensors entails the covalent and site-specific coupling of a bioluminescent sensor component to an analyte-specific antibody via protein G-mediated photoconjugation. Target detection is accomplished through intramolecular competition with a tethered analyte competitor for antibody binding. We established two variants of LUCOS: an inherent ratiometric LUCOSR variant and an intensiometric LUCOSI version, which can be used for ratiometric detection upon the addition of a split calibrator luciferase. To demonstrate the versatility of the LUCOS platform, sensors were developed for the detection of the small molecule cortisol and the protein biomarker NT-proBNP. Sensors for both targets displayed analytedependent changes in the emission ratio and enabled detection in the micromolar concentration range (K D,app = 16−92 μM). Furthermore, we showed that the response range of the LUCOS sensor can be adjusted by attenuating the affinity of the tethered NT-proBNP competitor, which enabled detection in the nanomolar concentration range (K D,app = 317 ± 26 nM). Overall, the LUCOS platform offers a highly versatile and easy method to convert commercially available monoclonal antibodies into bioluminescent biosensors that provide a homogeneous alternative for the competitive immunoassay.

Development of Luminescent Biosensors for Calprotectin

“…For the binders, we wanted to use a protein scaffold that is manufactured with ease to minimize the cost and complexity of the ultimate assay. We therefore chose to use Fabs (fragment antigen binding domains), which can be easily expressed in E. coli , and have been used to identify selective binders for a range of diverse antigen targets. − Moreover, Fabs have been engineered into a broad spectrum of biosensors, including, but not exclusively, antibody-based LUCIDs, pGA1-FAB LRT split-β-lactamase complementation assay, and Fab Q-body, demonstrating the versatility of Fabs as binding modules for biosensor development. We reasoned that the protease-resistant (S100A8/9) 2 heterotetramer form of calprotectin would be the desired target for sensor recognition.…”