Real-time, high-throughput measurements of peptide-MHC-I dissociation using a scintillation proximity assay.
Efficient presentation of peptide-MHC class I complexes to immune T cells depends upon stable peptide-MHC class I interactions. Theoretically, determining the rate of dissociation of a peptide-MHC class I complexes is straightforward; in practical terms, however, generating the accurate and closely timed data needed to determine the rate of dissociation is not simple. Ideally, one should use a homogenous assay involving an inexhaustible and label-free assay principle. Here, we present a homogenous, high-throughput peptide-MHC class I dissociation assay, which by and large fulfill these ideal requirements. To avoid labeling of the highly variable peptide, we labeled the invariant β2m and monitored its dissociation by a scintillation proximity assay, which has no separation steps and allows for real-time quantitative measurement of dissociation. Validating this work-around to create a virtually label-free assay, we showed that rates of peptide-MHC class I dissociation measured in this assay correlated well with rates of dissociation rates measured conventionally with labeled peptides. This assay can be used to measure the stability of any peptide-MHC class I combination, it is reproducible and it is well suited for high-throughput screening. To exemplify this, we screened a panel of 384 high-affinity peptides binding to the MHC class I molecule, HLA-A*02:01, and observed the rates of dissociation that ranged from 0.1h to 46h depending on the peptide used.
Laboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, Denmark. mikha@sund.ku.dk
Mikkel
M
Harndahl
Michael
M
Rasmussen
Gustav
G
Roder
Søren
S
Buus
D003199:Computer Systems; D015789:HLA-A2 Antigen / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D007158:Immunologic Techniques / Q000379:methods*; D007700:Kinetics; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D012588:Scintillation Counting / Q000379:methods; D001613:beta 2-Microglobulin / Q000378:metabolism
C435939:HLA-A*02:01 antigen; D015789:HLA-A2 Antigen; D010455:Peptides; D001613:beta 2-Microglobulin