Epitope testing using soluble HLA

Abstract

The present invention relates generally to a methodology for assaying the binding of a peptide to an individual, specific, soluble HLA molecule using fluorescence polarization. The peptides utilized in the method may be identified by indirect methods utilizing T lymphocytes, or by a direct method of epitope discovery described herein.

1 . A method of assaying a peptide for binding to an individual class I molecule, the method comprising the steps of: 
 providing a first peptide, wherein the first peptide is known to bind to a specific class I molecule;    labeling the first peptide;    providing individual soluble, specific class I molecules;    mixing the labeled first peptide with the individual soluble, specific class I molecules to provide individual soluble, specific class I molecule-labeled first peptide complexes;    isolating the individual soluble, specific class I molecule-labeled first peptide complexes;    providing a second peptide, wherein the second peptide is a peptide of interest;    mixing the second peptide with the individual soluble, specific class I molecule-labeled first peptide complexes; and    identifying individual soluble, specific class I molecule-second peptide complexes, wherein the second peptide has displaced the labeled first peptide and the individual soluble, specific class I molecule has the second peptide loaded therein.    
     
     
         2 . The method of  claim 1  wherein, in the step of labeling the first peptide, the first peptide is labeled with a radiolabel or a fluorescent label.  
     
     
         3 . The method of  claim 2  wherein, in the step of identifying individual soluble, specific class I molecule-second peptide complexes, the first peptide is labeled with a fluorescent label, and the individual soluble, specific class I molecule-second peptide complexes are identified by fluorescence polarization.  
     
     
         4 . The method of  claim 1  wherein, in the method of providing individual soluble, specific class I molecules, the individual soluble, specific class I molecules are produced by the method comprising the steps of: 
 obtaining genomic DNA or cDNA encoding at least one class I molecule;    identifying an allele encoding an individual class I molecule in the genomic DNA or cDNA;    PCR amplifying the allele encoding the individual class I molecule in a locus specific manner such that a PCR product produced therefrom encodes a truncated, soluble form of the individual class I molecule;    cloning the PCR product into an expression vector, thereby forming a construct that encodes the individual soluble, specific class I molecule;    transfecting the construct into a cell line to provide a cell line containing a construct that encodes an individual soluble, specific class I molecule, the cell line being able to naturally process proteins into peptide ligands capable of being loaded into antigen binding grooves of class I molecules;    culturing the cell line under conditions which allow for expression of the individual soluble, specific class I molecules from the construct, such conditions also allowing for endogenous loading of a peptide ligand into the antigen binding groove of each individual soluble, specific class I molecule prior to secretion of the individual soluble, specific class I molecules from the cell; and    isolating the secreted individual soluble, specific class I molecules.    
     
     
         5 . The method of  claim 4  wherein the construct further encodes a tag which is attached to the individual soluble, specific class I molecule and aids in isolating the individual soluble, specific class I molecule.  
     
     
         6 . The method of  claim 5  wherein the tag is selected from the group consisting of a HIS tail and a FLAG tail.