title: An improved assembly assay for peptide binding to HLA-B*2705 and H-2K(k) class I MHC molecules.
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 degrees 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.
authors.affiliation: The Nuffield Department of Clinical Medicine, John Radcliffe Hospital, Oxford, UK.
authors.forename: L
authors.initials: L
authors.lastname: Tan
authors.forename: M H
authors.initials: MH
authors.lastname: Andersen
authors.forename: T
authors.initials: T
authors.lastname: Elliott
authors.forename: J S
authors.initials: JS
authors.lastname: Haurum
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D000939:Epitopes; D006183:H-2 Antigens / Q000378:metabolism*; D015235:HLA-B Antigens / Q000378:metabolism*; D006801:Humans; D009842:Oligopeptides / Q000378:metabolism*; D010641:Phenotype; D011485:Protein Binding
chemical_list: D000939:Epitopes; D006183:H-2 Antigens; C024755:H-2K(K) antigen; D015235:HLA-B Antigens; D009842:Oligopeptides