| 主权项 |
1. A method for identifying at least one individual, endogenously loaded peptide ligand for an individual class I MHC molecule that distinguishes a transfected cell from a non-transfected cell, comprising the steps of:
culturing a non-transfected cell line and a transfected cell line, each containing a construct that encodes an individual soluble class I MHC molecule, and the non-transfected cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I MHC molecules, wherein the cell lines are cultured under conditions which allow for expression of the individual soluble class I MHC molecules from the construct, such conditions also allowing for endogenous loading of a peptide ligand in the antigen binding groove of each individual soluble class I MHC molecule prior to secretion of the individual soluble class I MHC molecules from the cell, and wherein the transfected cell line is produced by transfecting a non-transfected cell line with at least one of a gene from a microorganism and a tumor gene; isolating the secreted individual soluble class I MHC molecules having the endogenously loaded peptide ligands bound thereto from the non-transfected cell line and the transfected cell line; separating the endogenously loaded peptide ligands from the individual soluble class I MHC molecules from the non-transfected cell line and separating the endogenously loaded peptide ligands from the individual soluble class I MHC molecules from the transfected cell line; isolating the endogenously loaded peptide ligands from the non-transfected cell line and the endogenously loaded peptide ligands from the transfected cell line; comparing the endogenously loaded peptide ligands isolated from the transfected cell line to the endogenously loaded peptide ligands isolated from the non-transfected cell line; identifying at least one individual, endogenously loaded peptide ligand wherein:
(a) the peptide ligand is presented by the individual soluble class I MHC molecule on the transfected cell line that is not presented by the individual soluble class I MHC molecule on the non-transfected cell line; or(b) the peptide ligand is presented by the individual soluble class I MHC molecule on the non-transfected cell line that is not presented by the individual soluble class I MHC molecule on the transfected cell line; and identifying a source protein from which the at least one individual, endogenously loaded peptide ligand presented by the individual soluble class I MHC molecule on the transfected cell line and not presented by the individual soluble class I MHC molecule on the non-transfected cell line is obtained. |
