Methods of detecting alloantibodies to hla class ii antigens

Abstract

Described herein are materials and methods of incorporating CLIP peptide into the peptide binding groove of HLA Class II antigens and using such HLA Class II antigens for the detection of alloantibodies.

We claim: 
     
         1 . A method of preparing an HLA Class II antigen in which a Class II-associated invariant chain peptide (CLIP) is present in the peptide binding groove comprising:
 transfecting a host cell with a gene encoding an invariant chain and   expressing the HLA Class II antigen.   
     
     
         2 . The method of  claim 1  wherein the gene encoding the invariant chain is CD74. 
     
     
         3 . A method of preparing an HLA Class II antigen in which a CLIP peptide is present in the peptide binding groove above a selected threshold for determination of antibodies specific for HLA Class II antigens in which a CLIP is present in the peptide binding groove comprising:
 transfecting a host cell which expresses an invariant chain with a gene expressing an HLA Class II antigen.   
     
     
         4 .- 7 . (canceled) 
     
     
         8 . A method of screening for antibodies that specifically bind an HLA Class II antigen in which a CLIP is present in the peptide binding groove comprising the steps of:
 preparing a panel comprising a solid-phase substrate having an HLA Class II antigen in which a CLIP is present in the peptide binding groove immobilized or attached hereto;   obtaining a serum sample from a human subject,   contacting the panel with the serum sample, and   detecting binding of antibodies in said serum sample to the HLA Class II antigen in which a CLIP is present in the peptide binding groove.   
     
     
         9 . The method of  claim 8  wherein the HLA Class II antigen in which a CLIP is present in the peptide binding groove is prepared by the method of transfecting a host cell with a gene encoding an invariant chain and expressing the HLA Class II antigen. 
     
     
         10 . The method of  claim 8  wherein the HLA Class II antigen in which a CLIP is present in the peptide binding groove is prepared by the method of transfecting a host cell which expresses an invariant chain with a gene expressing an HLA Class II antigen. 
     
     
         11 . The method of  claim 8  wherein the HLA Class II antigen in which a CLIP is present in the peptide binding groove is prepared by the method of preparing an HLA Class II antigen and incubating said antigen with CLIP peptide under conditions selected to introduce said CLIP into the antigen peptide binding groove. 
     
     
         12 . The method of  claim 8  wherein antibodies that specifically bind an HLA Class II antigen in which the peptide binding groove is free of CLIP peptide are screened wherein a second solid-phase substrate is prepared having said HLA Class II antigen in which the peptide binding groove is free of CLIP above a selected threshold is contacted with the serum sample and binding of antibodies in the serum sample to the HLA Class II antigen in which the peptide binding groove is free of CLIP above a selected threshold is detected. 
     
     
         13 . The method of  claim 12  wherein the threshold is selected to avoid a false positive reaction. 
     
     
         14 . The method of  claim 11  wherein the solid-phase substrates are microparticles, wherein each microparticle presents at least one selected HLA antigen. 
     
     
         15 . The method of  claim 11  wherein binding of an antibody is detected using flow cytometry. 
     
     
         16 . The method of  claim 11  wherein antibody binding is detected with a secondary antibody. 
     
     
         17 . The method of  claim 11  wherein the secondary antibody comprises a label selected from the group consisting of a radioactive label, fluorescent label, enzymatic label, colorimetric label avidin label and biotin label. 
     
     
         18 . The method of  claim 11  wherein the human subject is a transplant or transfusion recipient. 
     
     
         19 . The method of  claim 11  wherein the human subject is a transplant or transfusion donor. 
     
     
         20 - 24 . (canceled)