Chaotropic Perturbation of Noncovalent Interactions of the Hemagglutinin Tag Monoclonal Antibody Fragment Enables Superresolution Molecular CensusClick to copy article linkArticle link copied!
- Hirushi GunasekaraHirushi GunasekaraDepartment of Chemistry, College of Liberal Arts and Sciences, University of Illinois at Chicago, Chicago, Illinois 60607-7061, United StatesMore by Hirushi Gunasekara
- Rangika MunaweeraRangika MunaweeraDepartment of Chemistry, College of Liberal Arts and Sciences, University of Illinois at Chicago, Chicago, Illinois 60607-7061, United StatesMore by Rangika Munaweera
- Ying S. Hu*Ying S. Hu*Email: [email protected]Department of Chemistry, College of Liberal Arts and Sciences, University of Illinois at Chicago, Chicago, Illinois 60607-7061, United StatesMore by Ying S. Hu
Abstract

Antibody–antigen interactions represent one of the most exploited biomolecular interactions in experimental biology. While numerous techniques harnessed immobilized antibodies for nanoscale fluorescence imaging, few utilized their reversible binding kinetics. Here, we investigated noncovalent interactions of the monoclonal hemagglutinin (HA) epitope tag antibody, 12CA5, in the fixed cellular environment. We observed that the use of a chaotropic agent, potassium thiocyanate (KSCN), promoted the dissociation of the 12CA5 antibody fragment (Fab), which already displayed faster dissociation compared to its immunoglobulin G (IgG) counterpart. Molecular dynamic simulations revealed notable root-mean-square deviations and destabilizations in the presence of KSCN, while the hydrogen-bonding network remained primarily unaffected at the antigen-binding site. The reversible interactions enabled us to achieve a superresolution molecular census of local populations of 3xHA tagged microtubule fibers with improved molecular quantification consistency compared to single-molecule localization microscopy (SMLM) techniques utilizing standard immunofluorescence staining for sample labeling. Our technique, termed superresolution census of molecular epitope tags (SR-COMET), highlights the utilization of reversible antibody–antigen interactions for SMLM-based quantitative superresolution imaging.
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