Methods and Systems for Detecting MHC Class I Binding Peptides

Abstract

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.

What is claimed is: 
     
         1 . A system comprising a solid surface, wherein the surface has attached thereto one or more MHC monomer or modified MHC monomer wherein the monomer denatures under denaturing conditions and reconstitutes to form a ternary complex containing a suitable MHC-binding peptide under reconstituting conditions. 
     
     
         2 . The system of  claim 1 , wherein the solid surface is a bead. 
     
     
         3 . The system of  claim 1 , wherein the solid surface is in a microtiter plate. 
     
     
         4 . The system of  claim 1 , wherein the solid surface is suitable for screening in a high throughput system. 
     
     
         5 . The system of  claim 1 , wherein attachment of the monomer to the solid surface is reversible. 
     
     
         6 . The system of  claim 1 , wherein attachment of the monomer to the solid surface is cleavable. 
     
     
         7 . The system of  claim 1 , wherein the solid surface is coated with a first binding ligand and the C-terminal end of the monomer is provided with a second binding ligand, wherein the first ligand binds specifically with the second ligand. 
     
     
         8 . The system of  claim 7 , wherein the first binding ligand is selected from avidin, streptavidin, neutravidin, StrepTactin and monomeric avidin and the second binding ligand is biotin. 
     
     
         9 . The system of  claim 8 , wherein the second binding ligand is attached to the monomer via a C-terminal end. 
     
     
         10 . The system of  claim 1 , wherein the denaturing conditions comprise a pH of about 2 to about 4. 
     
     
         11 . The system of  claim 1 , wherein the system further comprises an anti-MHC antibody that binds specifically to a conformational epitope that is present in the reconstituted monomer and absent in the denatured monomer. 
     
     
         12 . The system of  claim 1 , wherein the reconstituting conditions include a pH of from about 7 to about 8.5. 
     
     
         13 . The system of  claim 1 , wherein the system further comprises beta-2 microglobulin. 
     
     
         14 . The system of  claim 1 , wherein the monomer is HLA class I. 
     
     
         15 . The system of  claim 14  further comprising an anti-MHC class I monoclonal antibody, wherein the monoclonal antibody specifically binds to a reconstituted monomer and does not bind to a denatured monomer. 
     
     
         16 . The system of  claim 15 , wherein the monomer is HLA class I and the system further comprises beta-2 microglobulin and a suitable HLA-binding peptide of from about 8 to about 12 amino acids; wherein a reconstituted monomer binds to the beta-2 microglobulin and the suitable peptide under reconstituting conditions. 
     
     
         17 . The system of  claim 15 , wherein the monoclonal antibody is produced by hybridoma B9.12.1 
     
     
         18 . The system of  claim 1  or  16 , wherein the solid surface coated with the monomers is in a dried form. 
     
     
         19 . A kit comprising the system of  claim 1 ,  16  or  17 . 
     
     
         20 . The kit of  claim 19  further comprising an instruction. 
     
     
         21 . The kit of  claim 20  further comprising a control peptide to which the MHC monomer binds in a reconstituted form. 
     
     
         22 . A method for determining binding between a MHC monomer or modified MHC monomer and a putative MHC-binding peptide therefor, said method comprising:
 incubating under reconstituting conditions a solid surface having attached thereto a plurality of MHC monomers or modified MHC monomers in the presence and absence of the putative MHC-binding peptide, wherein the monomers have been denatured and reconstitute to form a ternary complex containing a suitable MHC-binding peptide under reconstituting conditions, and   determining binding to the MHC monomers after contact therewith of a monoclonal antibody that binds to the ternary complex but does not bind to dissociated components of the complex, which binding of the antibody indicates binding of the monomers with the putative MHC-binding peptide.   
     
     
         23 . The method of  claim 22 , wherein the denaturing conditions include a pH in the range from about 2 to about 4. 
     
     
         24 . The method of  claim 22 , further comprising separately incubating the monomers with a standard MHC-binding peptide for the monomers under the reconstituting conditions in the presence of the monoclonal antibody, and wherein the determining includes comparing binding of the antibody caused by the standard peptide to the binding of the antibody caused by the putative MHC-binding peptide. 
     
     
         25 . The method of  claim 24 , wherein the monomers are HLA class I, the monoclonal antibody is an anti-MHC-class I antibody, and the reconstituting conditions include the presence of sufficient beta-2 microglobulin for reconstitution of the monomers. 
     
     
         26 . The method of  claim 25 , wherein the monomers are HLA subclass A, B or C. 
     
     
         27 . The method of  claim 22 , wherein the monoclonal antibody is provided with a detectable label and the determining includes detecting the detectable label. 
     
     
         28 . The method of  claim 27 , wherein the detectable label is a secondary antibody that specifically binds to the monoclonal antibody. 
     
     
         29 . The method of  claim 28 , wherein the detectable label is fluorescent. 
     
     
         30 . The method of  claim 22 , wherein the solid surface is the wells of a microtiter plate or beads and the determining includes reading fluorescence with a fluorimeter. 
     
     
         31 . The method of  claim 30 , wherein the detecting further comprises detecting the fluorescence using high throughput scanning. 
     
     
         32 . The method of  claim 22 , wherein the solid surface is coated with avidin and the monomers are biotinylated to attach to the solid surface. 
     
     
         33 . The method of  claim 22 , wherein attachment of the monomers to the solid surface is reversible. 
     
     
         34 . The method of  claim 22 , wherein attachment of the monomers to the solid surface is cleavable. 
     
     
         35 . A method for determining the degree of binding affinity of an MHC monomer or modified MHC monomer for a putative MHC-binding peptide therefor, said method comprising:
 incubating at least one denatured MHC monomer or modified MHC monomer attached to a solid surface with the putative MHC-binding peptide and a monoclonal antibody that specifically binds to a conformational epitope in a ternary complex containing a corresponding reconstituted MHC monomer and does not bind to any dissociated component of the ternary complex, wherein the incubation is under reconstituting conditions; and   comparing binding of the monoclonal antibody to a ternary complex that contains the putative MHC-binding peptide with binding of the monoclonal antibody to a corresponding ternary complex containing the monomer and a known MHC-binding peptide,   
       wherein a difference in the bindings indicates the relative degree of binding affinity of the reconstituted monomer for the putative MHC-binding peptide. 
     
     
         36 . The method of  claim 35 , wherein the reconstituting conditions include a temperature in the range from about −18° C. to about 37° C. 
     
     
         37 . The method of  claim 35 , wherein the reconstituting conditions include a temperature in the range from about 4° C. to about 8° C. 
     
     
         38 . The method of  claim 35 , wherein the reconstituting conditions include a pH in the range from about 7 to about 8.5. 
     
     
         39 . The method of  claim 35 , wherein the reconstituting conditions include the presence of a suitable reconstitution buffer. 
     
     
         40 . The method of  claim 39 , wherein the suitable reconstitution buffer comprises polyoxyethylene(20) sorbitan monolaurate. 
     
     
         41 . The method of  claim 35 , wherein the incubating is for a period of from about 12 hours to about 48 hours. 
     
     
         42 . The method of  claim 35 , wherein the antibody is provided with a detectable label and wherein the comparison of binding comprises detecting a difference in the respective signals produced by the detectable label resulting from binding of the antibody thereto. 
     
     
         43 . The method of  claim 42 , wherein the antibody is labeled with a fluorescent label and the comparison of binding comprises detecting a difference in the respective fluorescence resulting from binding of the antibody thereto. 
     
     
         44 . The method of  claim 43 , wherein the solid surface is the wells of a microtiter plate or beads and the detecting includes reading the fluorescence with a fluorimeter. 
     
     
         45 . The method of  claim 44 , wherein the detecting further comprises detecting the fluorescence using a high throughput scanning. 
     
     
         46 . The method of  claim 35  wherein the fluorescent label is fluorescein isothiocyanate (FITC). 
     
     
         47 . The method of  claim 35 , wherein the monomers are HLA class I and further bind with beta-2 microglobulin in reconstituting conditions and the monoclonal antibody is an anti-MHC-class I monoclonal antibody. 
     
     
         48 . The method of  claim 47 , wherein the monomers are selected from HLA-A, HLA-B, and HLA C. 
     
     
         49 . The method of  claim 47 , wherein the monomers are chimeric. 
     
     
         50 . The method of  claim 35 , wherein the monoclonal antibody is produced by hybridoma B9.12.1. 
     
     
         51 . The method of  claim 42 , wherein the difference in the binding is compared after incubating the ternary complex containing the putative MHC binding peptide and the known peptide under conditions comprising a dissolution-testing temperature for a time sufficient to indicate the relative dissolution rate of the putative MHC-binding peptide. 
     
     
         52 . The method of  claim 51 , wherein the dissolution-testing temperature is in the range from about 4° C. to about 37° C. 
     
     
         53 . The method of  claim 52 , wherein the time is from about 2 hours to about 48 hours. 
     
     
         54 . The method of  claim 28 , wherein the detectable label is peroxidase.