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A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Published on: March 25, 2014
Real-time, high-throughput measurements of peptide-MHC-I dissociation using a scintillation proximity assay
Mikkel Harndahl1, Michael Rasmussen, Gustav Roder
1Laboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, Denmark. mikha@sund.ku.dk
Insights
We developed a novel, high-throughput assay to measure peptide-MHC class I dissociation rates. This label-free method accurately quantifies peptide-MHC stability, crucial for immune T cell interactions.
Area of Science:
- Immunology
- Biochemistry
- Molecular Biology
Background:
- Stable peptide-MHC class I interactions are essential for effective T cell immunity.
- Accurate measurement of peptide-MHC dissociation rates is challenging due to practical limitations in existing assays.
Purpose of the Study:
- To develop a homogenous, high-throughput, and virtually label-free assay for measuring peptide-MHC class I dissociation rates.
- To provide a reliable method for assessing the stability of diverse peptide-MHC class I complexes.
Main Methods:
- Developed a scintillation proximity assay by labeling the invariant β2m subunit of peptide-MHC class I complexes.
- Monitored β2m dissociation in real-time without requiring peptide labeling or separation steps.
- Validated the assay by comparing results with conventional methods using labeled peptides.
Main Results:
- The developed assay is homogenous, high-throughput, and avoids labeling of variable peptides.
- Dissociation rates measured using the novel assay correlated well with conventional methods.
- Screened 384 high-affinity peptides for HLA-A*02:01, revealing dissociation rates ranging from 0.1 to 46 hours.
Conclusions:
- The new assay accurately measures peptide-MHC class I dissociation rates and is suitable for high-throughput screening.
- This method offers a reproducible and efficient approach to evaluate peptide-MHC stability for various combinations.
- The assay facilitates a deeper understanding of peptide-MHC interactions critical for immune responses.
Abstract:
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.
