Affinity-matured recombinant antibody fragments analyzed by single-molecule force spectroscopy.
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.
Lehrstuhl für Angewandte Physik and Center for Nanoscience, Ludwig-Maximilians-Universität München, Munich, Germany. julia@morfill.de
Julia
J
Morfill
Kerstin
K
Blank
Christian
C
Zahnd
Beatrice
B
Luginbühl
Ferdinand
F
Kühner
Kay-E
KE
Gottschalk
Andreas
A
Plückthun
Hermann E
HE
Gaub
D001666:Binding Sites, Antibody; D001703:Biophysics / Q000379:methods*; D003001:Cloning, Molecular; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D018625:Microscopy, Atomic Force; D008956:Models, Chemical; D015233:Models, Statistical; D009154:Mutation; D012680:Sensitivity and Specificity; D013057:Spectrum Analysis / Q000379:methods*; D020349:Surface Plasmon Resonance
D007128:Immunoglobulin Fragments; D008024:Ligands