Methods of Detecting Antibodies Specific for Denatured HLA Antigens

Abstract

The invention is directed to methods of screening for HLA antibodies comprising detecting antibodies specific for native HLA antigens and denatured HLA antigens. The invention also provides for methods of removing antibodies specific for denatured HLA antigens or antibodies specific for native HLA antigens from a serum sample. In addition, the invention also provides for method of predicting whether a transplant recipient has an increased risk for rejecting the transplanted organ.

1 . A method of screening for antibodies that specifically bind a native HLA antigen, comprising the steps of:
 obtaining a serum sample from a human subject,   providing a first solid phase substrate having immobilized thereon said HLA antigen which has been denatured,   binding antibodies that specifically bind said denatured HLA antigen in said serum sample to said first solid-phase substrate to form an antibody/antigen complex,   separating the antibody/antigen complex from the serum sample to generate a serum sample essentially free of antibodies that specifically bind said denatured HLA antigen,   contacting the serum sample that is essentially free of antibodies that specifically bind said denatured HLA antigen with a second solid-phase substrate presenting the native HLA antigen, and   detecting binding of an antibody to the second solid-phase substrate, wherein binding of an antibody is indicative of a human subject having antibodies that specifically bind the native HLA antigen.   
     
     
         2 . A method of screening for antibodies that specifically bind a native HLA antigen, comprising the steps of:
 obtaining a serum sample from a human subject,   providing a cell that presents said HLA antigen which has been denatured,   binding antibodies that specifically bind said denatured HLA antigen in said serum sample to said cell to form an antibody/antigen complex,   separating the antibody/antigen complex from the serum sample to generate a serum sample essentially free of antibodies that specifically bind said denatured HLA antigen,   contacting the serum sample that is essentially free of antibodies that specifically bind said denatured HLA antigen with a solid-phase substrate presenting the native HLA antigen, and   detecting binding of an antibody to the solid-phase substrate presenting the native HLA antigen, wherein binding of an antibody is indicative of a human subject having antibodies that specifically bind the native HLA antigen.   
     
     
         3 . The method of  claim 1 , wherein the HLA antigen is a HLA class I antigen. 
     
     
         4 . The method  claim 1 , wherein the HLA antigen is a HLA class II antigen. 
     
     
         5 . The method of  claim 1 , wherein a solid-phase substrate is selected from microparticles, microbeads, magnetic beads and an affinity purification column. 
     
     
         6 . The method of  claim 1 , wherein the serum sample is obtained from a human subject that is a transplant or transfusion recipient. 
     
     
         7 . The method of  claim 1 , wherein the serum sample is obtained from a human subject that is a transplant or transfusion donor. 
     
     
         8 . A method of screening for antibodies that specifically bind a HLA antigen that has been denatured, comprising the steps of:
 obtaining a serum sample from a human subject,   providing a first solid-phase substrate having immobilized thereon said HLA antigen in its native conformation,   binding antibodies that specifically bind said native HLA antigen in said serum sample to said first solid-phase substrate to form an antibody/antigen complex,   separating the antibody/antigen complex from the serum sample to generate a serum sample essentially free of antibodies that specifically bind said native HLA antigen,   contacting the serum sample that is essentially free of antibodies that specifically bind said native HLA antigen with a second solid-phase substrate presenting the HLA antigen that has been denatured, and   detecting binding of an antibody to the second solid-phase substrate, wherein binding of an antibody is indicative of a human subject having antibodies that specifically bind the denatured HLA antigen.   
     
     
         9 . A method of screening for antibodies that specifically bind a HLA antigen that has been denatured, comprising the steps of:
 obtaining a serum sample from a human subject,   providing a cell that presents said HLA antigen in its native conformation,   binding antibodies that specifically bind said native HLA antigen in said serum sample to said cell to form an antibody/antigen complex,   separating the antibody/antigen complex from the serum sample to generate a serum sample essentially free of antibodies that specifically bind said native HLA antigen,   contacting the serum sample that is essentially free of antibodies that specifically bind said native HLA antigen with a solid-phase substrate presenting the HLA antigen that has been denatured, and   detecting binding of an antibody to the solid-phase substrate, wherein binding of an antibody is indicative of a human subject having antibodies that specifically bind the denatured HLA antigen.   
     
     
         10 . The method of  claim 8 , wherein the HLA antigen is a HLA class I antigen. 
     
     
         11 . The method  claim 8 , wherein the HLA antigen is a HLA class II antigen. 
     
     
         12 . The method of  claim 8 , wherein a solid-phase substrate is selected from microparticles, microbeads, magnetic beads and an affinity purification column. 
     
     
         13 . The method of  claim 8 , wherein the serum sample is obtained from a human subject that is a transplant or transfusion recipient. 
     
     
         14 . The method of  claim 8 , wherein the serum sample is obtained from a human subject that is a transplant or transfusion donor.