Thermofluorimetric Analysis (TFA) using Probes with Flexible Spacers: Application to Direct Antibody Sensing and to Antibody-Oligonucleotide (AbO) Conjugate Valency Monitoring

Antibodies have long been recognized as clinically relevant biomarkers of disease. The onset of a disease often stimulates antibody production at low quantities, making it crucial to develop sensitive, specific, and easy-to-use antibody assay platforms. Antibodies are also extensively used as probes in bioassays, and there is a need for simpler methods to evaluate specialized probes such as antibody-oligonucleotide (AbO) conjugates. Previously, we have demonstrated that thermofluorimetric analysis (TFA) of analyte-driven DNA assembly can be leveraged to detect protein biomarkers using AbO probes. A key advantage of this technique is its ability to circumvent autofluorescence arising from biological samples, which otherwise hampers homogenous assays. The analysis of differential DNA melt curves (dF/dT) successfully distinguishes the signal from background and interferences. Expanding the applicability of TFA further, here-in we demonstrate a unique proximity based TFA assay for antibody quantification which is functional in 90% human plasma. We show that conformational flexibility of the DNA-based proximity probes is critically important for optimal performance in these assays. To promote stable, proximity-induced hybridization of the short DNA strands, substitution of polyethylene glycol (PEG) spacers in place of ssDNA segments led to improved conformational flexibility and sensor performance. Finally, by applying these flexible spacers to study AbO conjugates directly, we validate this modified TFA approach as a novel tool to elucidate the probes valency, clearly distinguishing between monovalent and multivalent AbOs and reducing the reagent amounts by 12-fold