Abstract: The assembly assay for peptide binding to class I major histocompatibility complex (MHC) is based on the ability to stabilise MHC class I molecules from mutant cell lines by the addition of suitable peptides. Such cell lines lack a functional transporter associated with antigen presentation (TAP) and as a result accumulate empty, unstable class I molecules in the ER. These dissociate rapidly in cell lysates unless they are stabilised by the addition of an appropriate binding peptide during lysis. The extent of stabilisation of class I molecules is directly related to the binding affinity of the added peptide. However, some MHC class I molecules, including HLA-B*2705 and H-2Kk are unusually stable in their peptide-receptive state making them inappropriate for analysis using this assay or assays which depend on the ability of peptides to stabilise MHC class I molecules at the cell surface. Here we present an improved method that permits reliable measurements of peptide binding to such class I MHC molecules that are unusually stable in the absence of peptide. Cells are lysed in the presence of peptide and incubated at 4°C. After 2 h, during which peptide binding to empty MHC molecules occurs, the lysate is heated to a temperature which preferentially destabilises those MHC molecules that remain empty. We have used this technique to assay peptide binding to HLA-B*2705, as well as to the murine allele H-2Kk which also displays a stable phenotype when transfected into TAP-deficient T2 cells and show that this method represents a marked improvement over previous methods in terms of lower background signal and higher recovery of peptide bound molecules.

EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Istex/Checkpoint

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{{Explor lien
   |wiki=    Sante
   |area=    MersV1
   |flux=    Istex
   |étape=   Checkpoint
   |type=    RBID
   |clé=     ISTEX:A6D4925BB38A2541AA0035AA3BB812B456704254
   |texte=   An improved assembly assay for peptide binding to HLA-B*2705 and H-2Kk class I MHC molecules
}}