Measurement of Peptide Binding to MHC Class II Molecules by Fluorescence Polarization

Abstract

Peptide binding to major histocompatibility complex class II (MHCII) molecules is a key process in antigen presentation and CD4+ T cell epitope selection. This unit describes a fairly simple but powerful fluorescence polarization-based binding competition assay to measure peptide binding to soluble recombinant MHCII molecules. The binding of a peptide of interest to MHCII molecules is assessed based on its ability to inhibit the binding of a fluorescence-labeled probe peptide, with the strength of binding characterized as IC50 (concentration required for 50% inhibition of probe peptide binding). Data analysis related to this method is discussed. In addition, this unit includes a support protocol for fluorescence labeling peptide using an amine-reactive probe. The advantage of this protocol is that it allows simple, fast, and high throughput measurements of binding for a large set of peptides to MHCII molecules.

Evaluating the Role of HLA-DM in MHC Class II–Peptide Association Reactions

“…Fluorescence polarization (FP) assay was used to measure real time peptide association kinetics as described previously (53). Briefly, 25 nM Alexa488-labeled HA or CLIP was mixed with various concentrations of DR1 (ranging from 0.1 to 1.6 μM) in the presence of different DM concentrations (ranging from 0 to 1.6 μM) in 200 μl pH5.5 binding buffer (100 mM sodium citrate, 50 mM NaCl, 0.1% octylglucoside, 5 mM EDTA, 0.1% NaN3, 1 mM DTT, 1X protease inhibitor cocktail) in 96-well non-binding black polystyrene plates (Corning Incorporated, Corning, NY).…”

HLA-DO Modulates the Diversity of the MHC-II Self-peptidome

“…Binding Affinity and DM Sensitivity Measurements-For three abundant self-peptides, DM sensitivity has been previously characterized: CLIP, the invariant chain chaperone fragment efficiently removed by DM (18,54), DR␣ [52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68] , the human ortholog of the YAe epitope (55) known to be highly sensitive to DM exchange (56), and the transplantation alloepitope A2 104 -117 (39) previously demonstrated to be highly resistant to DM-mediated exchange (38). For these peptides, we summed the intensities of all peptides sharing the respective core epitopes and compared summed intensities between replicate samples of WT and DO-KO-1 cells.…”

Current tools for predicting cancer-specific T cell immunity

“…60 Alternatively, using insect cell expression systems, soluble "empty" MHC II molecules can be produced and peptide binding assessed by means of biotin or fluorochrome labeled peptides. 61,62 MHC II-peptide complex stability can be measured via the disappearance of the tagged peptide over time. Moreover, because MHC II molecules have an open-ended peptide-binding site, they can bind to immobilized peptides, allowing the use of peptide microarrays for MHC class II peptide screening by incubating these with "empty" class II molecules and detection with anti-MHC II mAb (Fig.…”