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Epitope Mapping of Antibody–Antigen Complexes by Nuclear Magnetic Resonance Spectroscopy

  • Osnat Rosen
  • Jacob AnglisterEmail author
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 524)

Summary

Nuclear magnetic resonance (NMR) is a very powerful tool for determining the boundaries of peptide epitopes recognized by antibodies. NMR can be used to study antibodies in complexes that exhibit a wide range of binding affinities from very weak and transient to very tight. Choice of the specific method depends upon the dissociation constant, especially the ligand off-rate.

Epitope mapping by NMR is based on the difference in mobility between the amino acid residues of a peptide antigen that interact tightly with the antibody and residues outside the epitope that do not interact with the antibody. The interacting peptide residues become considerably immobilized upon binding. Their mobility will resemble that of the antibody’s residues. Several NMR methods were developed based on these characteristics. In this chapter we discuss some of these methods, including dynamic filtering, comparison of 1H-15N HSQC peaks’ intensities, transverse relaxation time, measurements of 1H-15N nuclear Overhauser effect (NOE) values, and measurements of T relaxation time.

Key words

Epitope mapping NMR Antibody V3 gp120 Acetylcholine receptor a-bungarotoxin Dynamic filtering Relaxation times, Peptide antigen 

Notes

Acknowledgments

We thank Drs. Avraham Samson, Anat Zvi, Irina Kustanovic, Michal Sharon, and Naama Kessler, who did some of the studies described in this chapter. We gratefully acknowledge help from Dr. Tali Scherf in maintaining the NMR spectrometers and setting up some of the experiments. We thank Dr. Sandy Livnat for editorial assistance. This study was supported by the National Institute of Health Grant GM 53329 to Jacob Anglister who is the Dr. Joseph and Ruth Owades Professor of Chemistry.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Department of Structural BiologyWeizmann Institute of ScienceRehovotIsrael

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