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

02 February 2023, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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

Keywords

Thermofluorimetric analysis (TFA)
Antibodies
Antibody-oligonucleotide conjugates
polyethylene glycol spacers
Immunoassay

Supplementary materials

Title
Description
Actions
Title
Supporting Information for: Thermofluorimetric Analysis (TFA) using Probes with Flexible Spacers: Application to Direct Antibody Sensing and to Antibody-Oligonucleotide (AbO) Conjugate Valency Monitoring
Description
Supporting Information (SI) Contents: Page S-2: Sequences of DNA strands used in this study, Table S-1. Page S-3: Data analysis demonstration, Figure S-1. Page S-4: Gel images of antibody-oligo conjugates and related methods, Figure S-2. Pages S-5: Antibody-oligo valency studies, Figure S-3. Page S-6 : Methods used for thrombin detection by TFA. Page S-7: Thrombin sensing results, Figure S-4.
Actions

Comments

Comments are not moderated before they are posted, but they can be removed by the site moderators if they are found to be in contravention of our Commenting Policy [opens in a new tab] - please read this policy before you post. Comments should be used for scholarly discussion of the content in question. You can find more information about how to use the commenting feature here [opens in a new tab] .
This site is protected by reCAPTCHA and the Google Privacy Policy [opens in a new tab] and Terms of Service [opens in a new tab] apply.