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Peptide Binding to HLA Class I Molecules: Homogenous, High-Throughput Screening, and Affinity AssaysLaboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
Laboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
Laboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
Laboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark
Center for Biological Sequence Analysis, BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark
Laboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark, s.buus{at}immi.ku.dk 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 AlphaScreenTM). 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)
Key Words: high-throughput screening • peptide-HLA interaction • AlphaScreenTM • LOCI • homogeneous assay
This version was published on February
1, 2009 Journal of Biomolecular Screening, Vol. 14, No. 2,
173-180 (2009) |
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