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A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Published on: March 25, 2014
Functional recombinant MHC class II molecules and high-throughput peptide-binding assays
Sune Justesen1, Mikkel Harndahl, Kasper Lamberth
1Laboratory of Experimental Immunology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. sjust@sund.ku.dk
Immunome Research
|May 7, 2009
Summary
Researchers developed a new method to create recombinant major histocompatibility complex class II (MHC-II) molecules and assays for studying peptide binding. This advance facilitates the generation of MHC-II reagents for T helper cell research.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Major histocompatibility complex class II (MHC-II) molecules present exogenous peptides to CD4+ T helper cells.
- The high polymorphism of MHC-II presents challenges for peptide binding analysis and reagent generation.
- Existing methods for producing functional recombinant MHC-II and measuring peptide interactions are not standardized.
Purpose of the Study:
- To develop a robust methodology for generating recombinant MHC-II molecules.
- To establish assays for measuring peptide-MHC-II interactions.
- To create versatile MHC-II resources for immunological research.
Main Methods:
- Engineered MHC-II alpha and beta chains with modified transmembrane regions and a beta-chain biotinylation signal peptide.
- Expressed chains as inclusion bodies in E. coli, purified under denaturing conditions.
- Refolded chains with peptides and developed homogeneous, non-radioactive, high-throughput (HTS) binding assays.
Main Results:
- Efficient formation of peptide-MHC-II complexes was achieved through a refolding process.
- Developed HTS assays to determine peptide binding affinities, with best binders showing low nanomolar affinity.
- Successfully generated recombinant MHC-II molecules and HTS assays for nine different MHC-II types, including polymorphic variants.
Conclusions:
- Developed versatile recombinant MHC-II molecules and associated assays.
- These resources can aid in creating MHC class II-wide reagents, data, and tools.
- The method supports the study of diverse and polymorphic MHC-II molecules.
