A Signal Turn-On DNA Proximity Assay That Correlates with SARS-CoV-2 Blocking Antibody Levels

Surrogate in vitro assays have enabled the scalable testing of neutralizing antibodies to SARS-CoV-2 as a toolbox to manage the post-COVID-19 situation and future outbreaks. However, most surrogate assays use a competitive format to mimic the blocking event between the receptor binding domain (RBD) and the human angiotensin-converting enzyme 2 (ACE2) receptor. This signal "turn-off" format has a narrow dynamic range and is semiquantitative at best. In this proof-of-concept work, we introduce a signal "turn-on" dual antigen assay based on the proximity activation of a pair of RBD-oligonucleotide initiator probes upon bridging by the target antibody. Three well-characterized SARS-CoV-2 variants, i.e., 2019 (wild-type), Delta (B.1.617.2), and Omicron (B.1.1.529), were tested against three commercial antibodies with known RBD-binding and ACE2-blocking profiles. The DNA proximity assay (DPA) results correlated with the ability of the antibodies to block ACE2 instead of their binding to the RBD. Moreover, the single-step, wash-free property of DPA enabled quantification using the kinetic rate of fluorescence signal generation which was more robust than conventional end-point measurements. The DPA achieved good analytical performance with ng/mL detection limit and high assay precision (<5% interassay coefficient of variation), as well as good spike-and-recovery and dilution linearity in serum matrix. The test outcome from three sets of validated vaccine time-series samples were concordant with reference values, demonstrating the potential clinical utility of the DPA platform.