Ultra-low-abundance cancer biomarker discovery: Multiplexed protein profiling with attomolar sensitivity

Introduction: There is a great potential for utilizing ultra-low-abundance biomarkers in numerous applications across oncology research. These biomarkers, including many cancer-related signaling proteins and their post-translationally modified (PTM) isoforms, may provide key biological indicators, yet remain technically challenging to measure, particularly in multiplex. The leading discovery platforms available in the market lack the necessary sensitivity to reliably detect sub-fM biomarkers, and, therefore, do not include PTM isoforms in their panels. Based on published third-party assessments, 1,000+ plex platforms can return non-detects on over half of the targets in their panels for a given sample, due to insufficient sensitivity. Methods and Results: We have developed and validated a multiplex protein profiling technology, BlueSCAI™ ( B ackground L owering u sing S erial- C apture, A dapter- I nsertion), which can increase technical sensitivity down to the attomolar level. BlueSCAI is a proximity-based platform with a capture-release-recapture mechanism. We conducted a head-to-head comparison with an industry leading proximity profiling platform, encompassing an overlap of 21 randomly selected biomarkers, for which there were i. published LODs available for other commercial platform; and ii. the required reagents, and iii. the target antigens, were readily available from vetted vendors . Across numerous protein targets, BlueSCAI demonstrated limits of detection that are several logs lower than those of high plex protein platforms, representing a substantial improvement in analytical sensitivity. In addition, in comparisons with commercial ELISA assays, using the same antibody pairs, BlueSCAI demonstrated LODs almost 1,000 fold lower for multiple biomarkers. For example, BlueSCAI's integrated CA19-9 biomarker has an improved sensitivity of ~ 3 logs compared to CA19-9 measured using a leading clinical lab platform (<0.0001 U/mL vs 0.6 U/mL). Analysis of dose-response curves indicated that almost half of the target panel exhibited 4+ logs of linear dynamic range, with an equal fraction showing maximal signal above background of greater than 10,000. Cross-reactivity (1-signal specificity) was measured with a leave-out mixed antigen pool approach. Signal specificities exceeded 99.9% across ¾ of the target panel, and over 98% specific on the remaining 25% of targets. Conclusion: BlueSCAI's specific, high sensitivity, multiplex detection shows great promise for profiling new oncology-related, biologically informative, ultra-low-abundance biomarkers, which includes cancer signaling proteins, PTM isoforms, and extracellular vesicle cargo, across diverse cancer research applications.