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Affinity-Matured Recombinant Antibody Fragments Analyzed by Single-Molecule Force Spectroscopy

Julia Morfill ^*, Kerstin Blank ^*, Christian Zahnd , Beatrice Luginbühl , Ferdinand Kühner ^*, Kay-E. Gottschalk ^*, Andreas Plückthun  and Hermann E. Gaub ^*

^* Lehrstuhl für Angewandte Physik and Center for Nanoscience, Ludwig-Maximilians-Universität München, Munich, Germany; and Biochemisches Institut, Universität Zürich, Zürich, Switzerland

Correspondence: Address reprint requests to Julia Morfill, Lehrstuhl für Angewandte Physik and Center for Nanoscience, LMU München, Amalienstrasse 54, D-80799, Munich, Germany. Tel.: 49-89-2180-2306; Fax: 49-89-2180-2050; E-mail: julia{at}morfill.de.

For many applications, antibodies need to be engineered toward maximum affinity. Strategies are in demand to especially optimize this process toward slower dissociation rates, which correlate with the (un)binding forces. Using single-molecule force spectroscopy, we have characterized three variants of a recombinant antibody single-chain Fv fragment. These variants were taken from different steps of an affinity maturation process. Therefore, they are closely related and differ from each other by a few mutations only. The dissociation rates determined with the atomic force microscope differ by one order of magnitude and agree well with the values obtained from surface plasmon resonance measurements. However, the effective potential width of the binding complexes, which was derived from the dynamic force spectroscopy measurements, was found to be the same for the different mutants. The large potential width of 0.9 nm indicates that both the binding pocket and the peptide deform significantly during the unbinding process.

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