Antigen Binding Forces of Single Antilysozyme Fv Fragments Explored by Atomic Force Microscopy

Abstract

We used atomic force microscopy (AFM) to explore the antigen binding forces of individual Fv fragments of antilysozyme antibodies (Fv). To detect single molecular recognition events, genetically engineered histidine-tagged Fv fragments were coupled onto AFM tips modified with mixed self-assembled monolayers (SAMs) of nitrilotriacetic acid- and tri(ethylene glycol)-terminated alkanethiols while lysozyme (Lyso) was covalently immobilized onto mixed SAMs of carboxyl- and hydroxyl-terminated alkanethiols. The quality of the functionalization procedure was validated using X-ray photoelectron spectroscopy (surface chemical composition), AFM imaging (surface morphology in aqueous solution), and surface plasmon resonance (SPR, specific binding in aqueous solution). AFM force-distance curves recorded at a loading rate of 5000 pN/s between Fv- and Lyso-modified surfaces yielded a distribution of unbinding forces composed of integer multiples of an elementary force quantum of approximately 50 pN that we attribute to the rupture of a single antibody-antigen pair. Injection of a solution containing free Lyso caused a dramatic reduction of adhesion probability, indicating that the measured 50 pN unbinding forces are due to the specific antibody-antigen interaction. To investigate the dynamics of the interaction, force-distance curves were recorded at various loading rates. Plots of unbinding force vs log(loading rate) revealed two distinct linear regimes with ascending slopes, indicating multiple barriers were present in the energy landscape. The kinetic off-rate constant of dissociation (k(off) approximately = 1 x 10(-3) s(-1)) obtained by extrapolating the data of the low-strength regime to zero force was in the range of the k(off) estimated by SPR.

Specific Molecular Recognition and Nonspecific Contributions to Bacterial Interaction Forces

“…A 0.055-nN unbinding force was measured for protein A from the outer surface of a bacillus spore (31). Antigen binding of individual Fv fragments of antilysozyme antibodies (Fv) to lysozyme was accompanied by a single-bond interaction force of 0.050 nN (4).…”

XPS, TOF-SIMS, NEXAFS, and SPR Characterization of Nitrilotriacetic Acid-Terminated Self-Assembled Monolayers for Controllable Immobilization of Proteins

“…Examples include the quality of a pure NTA SAM 27 , conjugation chemistry for grafting NTA headgroups onto substrate surfaces 29 and the sequential surface activation step 28 . When combined with additional results from immunoassay 24,27 and molecular recognition experiments 32 , the bioactivity and orientation of proteins at solid/ liquid interfaces can be deduced. Surface structural information and NTA headgroup surface concentrations have not been addressed and related to protein specific immobilization and dissociation in flow assay conditions.…”

Lipid-dependent conformational dynamics underlie the functional versatility of T-cell receptor

“…In the conjugation experiment, we have made the density of the peptide attached to the cantilever as low as possible (10-fold less than the regular concentration) [53,54] and used 17.5 times molar excess mono-functional PEG to compete with bifunctional PEG to reduce multiple labeling of CD3ε CD peptide [55][56][57]. This protocol has been proven to be effective in single-molecule studies [56][57][58]. The peptides were chemically-synthesized with more than 95% purity.…”