A novel method to measure HLA-DM-susceptibility of peptides bound to MHC class II molecules based on peptide binding competition assay and differential IC(50) determination
Immunology & Microbiology
Program in Immunology and Microbiology; Department of Pathology; Department of Biochemistry and Molecular Pharmacology
Medical Subject Headings
Antibody Affinity; Antigen Presentation; Binding, Competitive; CD4-Positive T-Lymphocytes; Epitopes, T-Lymphocyte; Fluorescence Polarization; HLA-D Antigens; HLA-DR alpha-Chains; HLA-DRB1 Chains; Hemagglutinin Glycoproteins, Influenza Virus; Histocompatibility Antigens Class II; Humans; Inhibitory Concentration 50; Peptide Fragments; Protein Binding
Biochemistry | Immunology and Infectious Disease | Immunopathology
HLA-DM (DM) functions as a peptide editor that mediates the exchange of peptides loaded onto MHCII molecules by accelerating peptide dissociation and association kinetics. The relative DM-susceptibility of peptides bound to MHCII molecules correlates with antigen presentation and immunodominance hierarchy, and measurement of DM-susceptibility has been a key effort in this field. Current assays of DM-susceptibility, based on differential peptide dissociation rates measured for individually labeled peptides over a long time base, are difficult and cumbersome. Here, we present a novel method to measure DM-susceptibility based on peptide binding competition assays performed in the presence and absence of DM, reported as a delta-IC(50) (change in 50% inhibition concentration) value. We simulated binding competition reactions of peptides with various intrinsic and DM-catalyzed kinetic parameters and found that under a wide range of conditions the delta-IC(50) value is highly correlated with DM-susceptibility as measured in off-rate assay. We confirmed experimentally that DM-susceptibility measured by delta-IC(50) is comparable to that measured by traditional off-rate assay for peptides with known DM-susceptibility hierarchy. The major advantage of this method is that it allows simple, fast and high throughput measurement of DM-susceptibility for a large set of unlabeled peptides in studies of the mechanism of DM action and for identification of CD4+ T cell epitopes.
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Citation: J Immunol Methods. 2014 Apr;406:21-33. doi: 10.1016/j.jim.2014.02.008. Epub 2014 Feb 25. Link to article on publisher's site
Journal of immunological methods
Yin, Liusong and Stern, Lawrence J., "A novel method to measure HLA-DM-susceptibility of peptides bound to MHC class II molecules based on peptide binding competition assay and differential IC(50) determination" (2014). GSBS Student Publications. 1899.