AN IMPROVED ASSEMBLY ASSAY FOR PEPTIDE BINDING TO HLA-B-ASTERISK-2705AND H-2K(K) CLASS-I MHC MOLECULES

The assembly assay for peptide binding to class I major histocompatibi lity complex (MHC) is based on the ability to stabilise MHC class I mo lecules from mutant cell lines by the addition of suitable peptides. S uch cell Lines lack a functional transporter associated with antigen p resentation (TAP) and as a result accumulate empty, unstable class I m olecules in the ER. These dissociate rapidly in cell lysates unless th ey are stabilised by the addition of an appropriate binding peptide du ring lysis. The extent of stabilisation of class I molecules is direct ly related to the binding affinity of the added peptide. However, some MHC class I molecules, including HLA-B 2705 and H-2K(k) are unusuall y stable in their peptide-receptive state making them inappropriate fo r analysis using this assay or assays which depend on the ability of p eptides to stabilise MHC class I molecules at the cell surface. Here w e present an improved method that permits reliable measurements of pep tide binding to such class I MHC molecules that are unusually stable i n the absence of peptide. Cells are lysed in the presence of peptide a nd incubated at 4 degrees C. After 2 h, during which peptide binding t o empty MHC molecules occurs, the lysate is heated to a temperature wh ich 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-2K(k) which also displays a stable p henotype when transfected into TAP-deficient T2 cells and show that th is method represents a marked improvement over previous methods in ter ms of lower background signal and higher recovery of peptide bound mol ecules. (C) 1997 Elsevier Science B.V.