UNIT 18.3 Measurement of MHC/Peptide Interactions by Gel Filtration
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Immunology
How to Cite
Sidney, J., Southwood, S., Oseroff, C., del Guercio, M.-F., Sette, A. and Grey, H. M. 2001. Measurement of MHC/Peptide Interactions by Gel Filtration. Current Protocols in Immunology. 31:18.3:18.3.1–18.3.19.
- Published Online: 1 MAY 2001
- Published Print: JUN 1999
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This unit describes a technique for the direct and quantitative measurement of the capacity of peptide ligands to bind Class I and Class II MHC molecules. The binding of a peptide of interest to MHC is assessed based on its ability to inhibit the binding of a radiolabeled probe peptide to MHC molecules. The establishment of an MHC/peptide binding assay, and its subsequent use in determining the MHC binding capacities of peptide ligands, requires sufficient stocks of purified MHC and both labeled and unlabeled peptides. Accordingly, this unit includes protocols for the purification of Class I and Class II MHC molecules by affinity chromatography, and for the radiolabeling of peptides using the chloramine T method. A support protocol describes alterations in the that are necessary when performing direct binding assays, which are required for (1) selecting appropriate high-affinity, assay-specific, radiolabeled ligands and (2) determining the amount of MHC necessary to yield assays with the highest sensitivity. After a 2-day incubation, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Two methods for separation by size-exclusion gel-filtration chromatography are described, as is data analysis.