Peptide Binding to HLA Class I Molecules: Homogenous, High-Throughput Screening, and Affinity Assays

Abstract

The Human MHC Project aims at large-scale description of peptide-HLA binding to a wide range of HLA molecules covering all populations of the world and the accompanying generation of bioinformatics tools capable of predicting binding of any given peptide to any given HLA molecule. Here, the authors present a homogenous, proximity-based assay for detection of peptide binding to HLA class I molecules. It uses a conformation-dependent anti-HLA class I antibody, W6/32, as one tag and a biotinylated recombinant HLA class I molecule as the other tag, and a proximity-based signal is generated through the luminescent oxygen channeling immunoassay technology (abbreviated LOCI and commercialized as AlphaScreen). Compared with an enzyme-linked immunosorbent assay-based peptide-HLA class I binding assay, the LOCI assay yields virtually identical affinity measurements, although having a broader dynamic range, better signal-to-background ratios, and a higher capacity. They also describe an efficient approach to screen peptides for binding to HLA molecules. For the occasional user, this will serve as a robust, simple peptide-HLA binding assay. For the more dedicated user, it can easily be performed in a high-throughput screening mode using standard liquid handling robotics and 384-well plates. We have successfully applied this assay to more than 60 different HLA molecules, leading to more than 2 million measurements. (Journal of Biomolecular Screening 2009:173-180)

Tapasin Discriminates Peptide-Human Leukocyte Antigen-A*02:01 Complexes Formed with Natural Ligands

“…The affinities of the same HLA-A*02:01 binding peptides on the HLA-A*02: . The mixtures were incubated at 18°C for 48 h, and folded peptide-HLA-I complexes were detected by the HLA-I conformation-specific W6/32 monoclonal antibody in a homogenous assay (36). B, a study of Tpn 1-87 facilitation based on the maximum amount of folded peptide-HLA-I complexes, B max .…”

“…HLA-I Folding Assay-The peptide-HLA-I folding assays were done as described previously (13,36). Briefly, 2 nM of biotinylated recombinant HLA-I HC was diluted into a mixture of peptide and 30 nM recombinant human ␤ 2 m in the absence or presence of 20 nM Tpn in PBS containing 50 mM Tris and maleic acid, pH 6.6.…”

Uncovering the Peptide-Binding Specificities of HLA-C: A General Strategy To Determine the Specificity of Any MHC Class I Molecule

“…Multiplying the relevant RB values of the different amino acids in the different positions, we predicted the binding of each peptide/HLA-C allotype combination. For each HLA-C allotype, we then ranked the peptides, selected the 100 highest scoring peptides, and measured the affinity of binding of each peptide using a previously published high-throughput peptide-binding assay (25). Eight HLA-C allotypes were included in this analysis (i.e., HLA-C*03:03, -C*04:01, -C*05:01, -C*06:02, -C*07:02, -C*12:03, -C*14:02 and -C*15:02).…”

“…Peptide affinities of HLA-C molecules were determined using a luminescent oxygen channeling immunoassay (25). Briefly, peptides were dissolved in PBS/0.1% Lutrol F68 by sonication for 10 min.…”

Modified Human Beta 2‐Microglobulin (desLys58) Displays Decreased Affinity for the Heavy Chain of MHC Class I and Induces Nitric Oxide Production and Apoptosis

“…b binding OVA 257-264 peptide (SIINFEKL) and an HLA-A2 binding HBVc [18][19][20][21][22][23][24][25][26][27] peptide (FLPSDYFPSV) were synthesized by Shafer-N (Copenhagen, Denmark). Peptides were dissolved in distilled water, freeze-dried and stored at )20°C.…”