Methods and systems for detecting MHC class I binding peptides

摘要

The present invention is based on the discovery that MHC heavy chain monomers immobilized to a solid surface are still capable of forming detectable conformational epitopes and being detected by conformation-dependent antibodies. Methods for detecting peptide binding to HLA monomers, and methods for measuring the relative degree of binding between two MHC-binding peptides as well as a method of measurement for the rate of dissociation of peptides from MHC complexes are provided. The present invention also provides systems and kits useful for conducting the methods of the present invention.

主权项

1. A method to determine binding of a putative MHC binding peptide in a ternary MHC complex with MHC monomer or modified MHC monomer, the method comprising
(a) exposing a first ternary MHC complex comprising a MHC monomer or modified MHC monomer, peptide, and β2-microglobulin to denaturing conditions comprising a pH of about 2 to about 4 to dissociate the peptide and β2-microglobulin from the MHC monomer or modified MHC monomer to provide a denatured MHC monomer or modified MHC monomer, wherein the MHC monomer or modified MHC monomer is biotinylated and attached to a solid surface comprising avidin or streptavidin through a binding interaction between biotin and the avidin or streptavidin and the denatured MHC monomer or denatured modified MHC monomer remains attached to the solid surface; (b) washing the solid surface; (c) exposing, under renaturing conditions, the denatured MHC monomer or modified MHC monomer attached to the solid surface to a solution comprising β2-microglobulin and the putative MHC binding peptide; (d) determining formation of a second ternary MHC complex comprising the MHC monomer or modified MHC monomer, β2-microglobulin and the putative MHC binding peptide on the solid surface by contacting with an antibody that binds to a ternary MHC complex but not denatured MHC monomer or modified MHC monomer, and detecting binding of the antibody, wherein binding of the antibody indicates formation of the second ternary MHC complex comprising the putative peptide and that the putative MHC binding peptide comprises binding affinity for the MHC monomer or modified MHC monomer.