Studying MHC class II peptide loading and editing in vitro

Aeryon Kim, Isabel Ishizuka, Isamu Hartman, Yuri Poluektov, Kedar Narayan, Scheherazade Sadegh-Nasseri

Research output: Chapter in Book/Report/Conference proceedingChapter

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 fl uorescently 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.

Original languageEnglish (US)
Title of host publicationAntigen Processing
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages447-459
Number of pages13
ISBN (Print)9781627032179
DOIs
StatePublished - 2013

Publication series

NameMethods in Molecular Biology
Volume960
ISSN (Print)1064-3745

Keywords

  • BIAcore surface plasmon resonance
  • Fluorescent peptide
  • Gentle SDS-PAGE assay
  • HLA-DM editing
  • Kinetic studies
  • Peptide binding to HLA-DR
  • Tryptophan fluorescence assay

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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