Abstract
HLA-DM is now known to have a major contribution to the selection of immunodominant epitopes. A better understanding of the mechanisms controlling epitope selection can be achieved by examination of the biophysical behavior of major histocompatibility complex (MHC) class II molecules upon binding of antigenic peptides and the effect of DM on the interactions. Using purified soluble molecules, in this chapter, we describe several in vitro methods for measuring peptide binding to HLA-DR molecules and the effects of HLA-DM on the interactions. A simple qualitative method, Gentle SDS-PAGE Assay, would assess the ability of peptides to form tight complexes with MHC class II molecules. Measuring binding kinetics is among the most informative approaches to understanding molecular mechanisms, and here we describe two different methods for measuring binding kinetics of peptide–MHC complexes. In one method, rates of association and dissociation of fluorescently labeled peptides to soluble MHC class II molecules can be determined using G50 spin columns to separate unbound peptides from those in complex with MHC molecules. In another method, association and dissociation of unlabeled peptides and MHC class II molecules can be determined in real time using BIAcore surface plasmon resonance (SPR). We also have described an Intrinsic Tryptophan Fluorescence Assay for studying transient interactions of DM and MHC class II molecules.
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References
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Acknowledgements
This work was supported by NIH R01 grants AI063764 and R56AI091923 to S.S.-N., and a US National Science Foundation predoctoral award to I.Z.H. A.K. was a recipient of T32 grants to Graduate Immunology and Rheumatology programs during 2008–2011.
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Kim, A., Ishizuka, I., Hartman, I., Poluektov, Y., Narayan, K., Sadegh-Nasseri, S. (2013). Studying MHC Class II Peptide Loading and Editing In Vitro. In: van Endert, P. (eds) Antigen Processing. Methods in Molecular Biology™, vol 960. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-218-6_33
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DOI: https://doi.org/10.1007/978-1-62703-218-6_33
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