Antigen binding forces of individually addressed single-chain Fv antibody molecules
Robert Ros,Falk Schwesinger,Dario Anselmetti,Martina Kubon,Rolf Schäfer,Andreas Plückthun,Louis Tiefenauer +6 more
TLDR
This strategy of covalent immobilization and measuring well separated single molecules allows the characterization of ligand binding forces in molecular repertoires at the single molecule level and will provide a deeper insight into biorecognition processes.Abstract:
Antibody single-chain Fv fragment (scFv) molecules that are specific for fluorescein have been engineered with a C-terminal cysteine for a directed immobilization on a flat gold surface. Individual scFv molecules can be identified by atomic force microscopy. For selected molecules the antigen binding forces are then determined by using a tip modified with covalently immobilized antigen. An scFv mutant of 12% lower free energy for ligand binding exhibits a statistically significant 20% lower binding force. This strategy of covalent immobilization and measuring well separated single molecules allows the characterization of ligand binding forces in molecular repertoires at the single molecule level and will provide a deeper insight into biorecognition processes.read more
Citations
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Detection and localization of single molecular recognition events using atomic force microscopy
TL;DR: The current methodology for molecular recognition studies using the AFM is described, with an emphasis on strategies available for preparing AFM tips and samples, and on procedures for detecting and localizing single molecular recognition events.
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Micromechanical cantilever-based biosensors
TL;DR: In this paper, the authors summarize how cantilever-based sensors can be operated, and their working principle is presented in few selected biosensing experiments which have been performed recently in our groups in the study of biotin-streptavidin and antigen-antibody interactions, and specific surface charge development of organic molecules.
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Dynamic force spectroscopy of single DNA molecules
TL;DR: The forces to mechanically separate a single DNA duplex under physiological conditions by pulling at the opposite 5'-ends as a function of the loading rate are measured with an atomic force microscope to explore the analytic relevance of unbinding force measurements between complementary DNA strands.
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Single-molecule chemistry
TL;DR: The scanning tunneling microscope (STM) as discussed by the authors is a state-of-the-art system for direct visualization of chemistry by revealing the fundamental properties of atoms and molecules and their interactions with each other and the environment.
Journal ArticleDOI
Force Spectroscopy of Molecular Systems-Single Molecule Spectroscopy of Polymers and Biomolecules.
TL;DR: The intention of this review article is to present a critical evaluation of the actual development of static force spectroscopy, and mainly focuses on experiments dealing with inter- and intramolecular forces-starting with "simple" electrostatic forces, then ligand-receptor systems, and finally the stretching of individual molecules.
References
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Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library.
Tristan J. Vaughan,Andrew James Williams,Kevin Pritchard,Jane K. Osbourn,Anthony Richard Pope,John C. Earnshaw,John McCafferty,Regina A. Hodits,Jane Wilton,Kevin Stuart Johnson +9 more
TL;DR: This work shows that conventional hybridoma technology may be superseded by large phage libraries that are proving to be a stable and reliable source of specific, high affinity human monoclonal antibodies.
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Detection and localization of individual antibody-antigen recognition events by atomic force microscopy
TL;DR: It is indicated that this methodology has promise for characterizing rate constants and kinetics of molecular recognition complexes and for molecular mapping of biosurfaces such as membranes.
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Single Molecule Force Spectroscopy on Polysaccharides by Atomic Force Microscopy
TL;DR: Dextran filaments linked to a gold surface were probed with the atomic force microscope tip by vertical stretching and the conformational change was found to be reversible and was corroborated by molecular dynamics calculations.