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Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy
Published on: August 20, 2018
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Affinity-matured recombinant antibody fragments analyzed by single-molecule force spectroscopy
Julia Morfill1, Kerstin Blank, Christian Zahnd
1Lehrstuhl für Angewandte Physik and Center for Nanoscience, Ludwig-Maximilians-Universität München, Munich, Germany. julia@morfill.de
Biophysical Journal
|August 7, 2007
Summary
Researchers optimized antibody affinity by studying binding forces. Despite mutations, the binding pocket deformation remained consistent, revealing insights into antibody-antigen interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Antibody affinity is crucial for many applications.
- Optimizing antibody dissociation rates is key for enhanced binding.
- Understanding unbinding forces provides insights into molecular interactions.
Purpose of the Study:
- To characterize antibody variants engineered for maximum affinity.
- To investigate the relationship between mutations, dissociation rates, and binding forces.
- To determine the effective potential width of antibody-antigen binding complexes.
Main Methods:
- Single-molecule force spectroscopy (SMFS) was employed.
- Atomic force microscopy (AFM) was used to measure dissociation rates.
- Dynamic force spectroscopy (DFS) provided insights into binding complex potential width.
Main Results:
- Dissociation rates varied by an order of magnitude across antibody variants.
- AFM-measured dissociation rates correlated well with surface plasmon resonance (SPR) data.
- The effective potential width of binding complexes remained constant (0.9 nm) for all variants.
Conclusions:
- Antibody affinity maturation can be effectively studied using force spectroscopy.
- Significant deformation of the binding pocket and peptide occurs during unbinding.
- The consistent potential width suggests a conserved unbinding mechanism despite affinity changes.
