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. 2013 Feb:Chapter 18:Unit 18.3..
doi: 10.1002/0471142735.im1803s100.

Measurement of MHC/peptide interactions by gel filtration or monoclonal antibody capture

John Sidney  1 , Scott Southwood, Carrie Moore, Carla Oseroff, Clemencia Pinilla, Howard M Grey, Alessandro Sette
Affiliations

Measurement of MHC/peptide interactions by gel filtration or monoclonal antibody capture

John Sidney et al. Curr Protoc Immunol. 2013 Feb.

Abstract

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 purified MHC molecules. 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. An alternate protocol describes alterations in the basic protocol 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 predetermined incubation period, dependent upon the allele under examination, the bound and unbound radiolabeled species are separated, and their relative amounts are determined. Three methods for separation are described, two utilizing size-exclusion gel-filtration chromatography and a third using monoclonal antibody capture of MHC. Data analysis for each method is also explained.

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Figures

Figure 18.3.1
Figure 18.3.1
A schematic overview of the steps involved in performing an MHC-peptide binding assay.
Figure 18.3.2
Figure 18.3.2
Example layout for serial dilution of inhibitor peptides amenable to high throughput screening.
Figure 18.3.3
Figure 18.3.3
Example layout for setting-up assays plates to screen serial dilutions covering a 6-log dose range.
See this image and copyright information in PMC

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References

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