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

Mikkel Harndahl, Sune Frederik Lamdahl Justesen, Kasper Lamberth, Gustav Røder, Morten Nielsen, Søren Buus

    Research output: Contribution to journalJournal articleResearchpeer-review

    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 (TM)). 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)
    Original languageEnglish
    JournalJournal of Biomolecular Screening
    Volume14
    Issue number2
    Pages (from-to)173-180
    ISSN1087-0571
    DOIs
    Publication statusPublished - 2009

    Keywords

    • homogeneous assay
    • high-throughput screening
    • peptide-HLA interaction
    • AlphaScreen (TM)
    • LOCI

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