Antigen binding forces of individually addressed single-chain Fv antibody molecules

Antigen binding forces of individually addressed single-chain Fv antibody molecules

^*Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland; ^‡Institute of Biochemistry, University of Zürich, CH-8057 Zurich, Switzerland; ^§Novartis Services Ltd., CH-4002 Basel, Switzerland; and ^¶Institute of Physics, University of Basel, CH-4056 Basel, Switzerland

Communicated by Hans Frauenfelder, Los Alamos National Laboratory, Los Alamos, NM (received for review February 8, 1998)

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.

Footnotes

↵ † R.R. and F.S. contributed equally to this work.
↵ ‖ To whom reprint requests should be addressed. e-mail: tiefenauer{at}psi.ch.
ABBREVIATIONS:

AFM,

atomic force microscopy;

scFv,

single-chain Fv;

VH and VL,

variable domains of heavy and light immunoglobulin chains