Peptide binding to HLA class I molecules: homogenous, high-throughput screening, and affinity assays

Area of Science:

• Immunology
• Biochemistry
• Bioinformatics

Background:

• The Human MHC Project seeks to map peptide-HLA binding across global populations.
• Accurate peptide-HLA binding data is crucial for understanding immune responses and developing predictive tools.

Purpose of the Study:

• To present a novel, homogenous, proximity-based assay for detecting peptide binding to HLA class I molecules.
• To offer a robust and scalable method for peptide-HLA binding screening.

Main Methods:

• Utilized Luminescent Oxygen Channeling Immunoassay (LOCI) technology, also known as AlphaScreen.
• Employed a conformation-dependent anti-HLA class I antibody (W6/32) and a biotinylated recombinant HLA class I molecule.
• Developed a proximity-based signal generation system.

Main Results:

• The LOCI assay demonstrated comparable affinity measurements to traditional ELISAs but with a broader dynamic range and improved signal-to-background ratios.
• Successfully applied the assay to over 60 HLA molecules, generating more than 2 million measurements.
• The assay is adaptable for both occasional users and high-throughput screening.

Conclusions:

• The LOCI assay provides a reliable, efficient, and scalable method for measuring peptide-HLA class I binding.
• This assay facilitates large-scale data generation for the Human MHC Project and aids in the development of predictive bioinformatics tools.