Non-radioactive detection of MHC class II-peptide antigen complexes in the sub-picomole range by high-performance size-exclusion chromatography with fluorescence detection.
In order to avoid chemical or structural modification of T-cell epitopes by labelling, a high-performance size-exclusion chromatographic fluorescence binding assay was developed, based on the intrinsic Trp fluorescence of major histocompatibility complex (MHC) proteins. The increase in Trp fluorescence intensity of the isolated human MHC product HLA-DR 1 on complex formation with unlabelled influenza matrix peptide[18-29] (IM[18-29]) was examined. Binding of IM[18-29] to the heterodimeric form of HLA-DR 1 (Kd = 4.8 mM) and to the disassembled alpha-and beta-subunits (Kd = 9.2 mM) could be demonstrated. In addition, the assay showed the peptide-induced formation of a dimeric conformer of HLA-DR 1, the nature of which is still undefined. Detection of HLA-DR 1 subunit-peptide complexes was possible in amounts of 25 ng in 10 microliter (80 fmol/microliter). The technique proved to be reproducible and less time consuming than common methods that need fluorescence or radioactive labelling.
Kalbacher
H
H
Medizinisch-naturwissenschaftliches Forschungszentrum der Universität Tübingen, Germany.
Kropshofer
H
H
D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006684:HLA-DR Antigens / Q000032:analysis* / Q000276:immunology; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D010455:Peptides / Q000032:analysis* / Q000276:immunology; D013050:Spectrometry, Fluorescence
D006684:HLA-DR Antigens; D007202:Indicators and Reagents; D010455:Peptides