Duplex detection of anti-drug antibodies achieved by a spectrally resolved bioluminescence platform

Bioluminescence (BL)-based biosensor represents a valuable and versatile analytical tool which offers high detection sensitivity and allows a streamlined, homogeneous workflow. However, nearly all existing BL assays are operated in a single-channel mode, which limits the detection capacity to one analyte at a time. This limitation largely restricts the application of BL assays in multiplex detection of biomarkers or anti-drug antibodies (ADAs). In this study, we developed the Dual-SplitNluc platform, a spectrally resolved duplex BL reporter system which enables duplex detection of two analytes in homogeneous format based on BL spectral unmixing. Dual-SplitNluc integrates our previously reported SplitNluc system with SplitNanoBRET, an orange-emitting complementation-based immunoassay platform derived from SplitNluc. With chemical modification of the SplitNluc reporter by adding a fluorophore serving as a BL resonance energy transfer acceptor, red-shifted photon emission can be generated and spectrally separated from the conventional SplitNluc signal. Through mathematical deconvolution of the spectrally integrated luminescence readouts, the signal spillover can be eliminated to recover the original BL signals from each channel, enabling ratiometric or quantitative analysis of both analytes. As a proof-of-concept study, we demonstrated the utilization of Dual-SplitNluc for simultaneous measurement of ADAs against two distinct targets. With spectral deconvolution strategy, the assay achieved high precision of ADA quantification of broad dynamic ranges. We further demonstrated the potential of Dual-SplitNluc in duplex neutralizing antibody detection. Collectively, Dual-SplitNluc extends BL assays beyond the single-analyte mode and provides a generalizable strategy for duplex and potentially higher-order multiplex homogeneous detection using spectral resolution as an orthogonal analytical dimension.