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
Name:
Publisher version
View Source
Access full-text PDFOpen Access
View Source
Check access options
Check access options
Student Authors
Liusong YinUMass Chan Affiliations
Department of Biochemistry and Molecular PharmacologyDepartment of Pathology
Program in Immunology and Microbiology
Document Type
Journal ArticlePublication Date
2014-04-01Keywords
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 BindingBiochemistry
Immunology and Infectious Disease
Immunopathology
Metadata
Show full item recordAbstract
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.Source
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 siteDOI
10.1016/j.jim.2014.02.008Permanent Link to this Item
http://hdl.handle.net/20.500.14038/33374PubMed ID
24583195Related Resources
Link to Article in PubMedae974a485f413a2113503eed53cd6c53
10.1016/j.jim.2014.02.008