Methods and kits for measuring ADAMTS13/FXI complexes

Abstract

Provided are assays and kits to detect ADAMTS13 activity using peptide substrates and ADAMTS13/Factor XI complexes using ELISA. These assays and kits can be used for diagnostic applications and to evaluate treatment of thrombotic thrombocytopenic purpura (TTP). Also provided is a novel form of ADAMTS13 found on platelets.

1 . A method for detecting ADAMTS13/FXI complexes in a sample comprising: 
 a) binding an anti-ADAMTS13 antibody to a solid phase;    b) adding the sample to the solid phase, wherein ADAMTS13/FXI complexes present in the sample binds to the antibody;    c) adding an anti-FXI antibody to the solid phase, wherein the FXI antibody binds to FXI in ADAMTS13/FXI complexes; and    d) detecting ADAMTS13/FXI complexes by direct or indirect immunolabelling of the anti-FXI antibody.    
     
     
         2 . The method of  claim 1 , wherein the sample is selected from the group consisting of a biological fluid, whole blood, plasma, platelet rich plasma (PRP), platelet poor plasma (PPP), pooled normal plasma (PNP), washed platelets and tissue culture supernatant.  
     
     
         3 . The method of  claim 1 , wherein the solid phase is a membrane, plate, microwell or bead.  
     
     
         4 . The method of  claim 1 , wherein detecting ADAMTS13/FXI complexes is by direct immunolabelling of the anti-FXI antibody.  
     
     
         5 . The method of  claim 4 , wherein the anti-FXI antibody is labeled with horse radish peroxidase.  
     
     
         6 . The method of  claim 1 , wherein detecting ADAMTS13/FXI complexes is by indirect immunolabelling of the anti-FXI antibody.  
     
     
         7 . The method of  claim 6 , wherein the anti-FXI antibody is raised in a first species and wherein a labeled antibody raised in a second species against antibodies from the first species is added to the solid phase and detected.  
     
     
         8 . A method for measuring the amount of ADAMTS13/FXI complexes in a sample comprising the method of  claim 1  and further comprising: 
 e) quantifying anti-FXI antibody detected, thereby measuring the amount of ADAMTS13/FXI complexes in the sample.    
     
     
         9 . A method for diagnosing thrombotic thrombocytopenia purpura (TTP) in a subject comprising 
 a) measuring the amount of ADAMTS13/FXI complexes in a test sample from the subject according to the method of  claim 8;  and    b) comparing the amount of ADAMTS13/FXI complexes in the test sample to the amount of ADAMTS13/FXI complexes in a control sample having normal ADAMTS13 activity;    wherein TTP is diagnosed by a reduced amount of ADAMTS13/FXI complexes in the test sample compared with the control sample.    
     
     
         10 . The method of  claim 9 , wherein the test sample is selected from the group consisting of whole blood, plasma, platelet rich plasma (PRP), platelet poor plasma (PPP), pooled normal plasma (PNP) and washed platelets.  
     
     
         11 . The method of  claim 9 , wherein the solid phase is a membrane, plate, microwell or bead.  
     
     
         12 . The method of  claim 9 , wherein detecting ADAMTS13/FXI complexes is by direct immunolabelling of the anti-FXI antibody.  
     
     
         13 . The method of  claim 12 , wherein the anti-FXI antibody is labeled with horse radish peroxidase.  
     
     
         14 . The method of  claim 9 , wherein detecting ADAMTS13/FXI complexes is by indirect immunolabelling of the anti-FXI antibody.  
     
     
         15 . The method of  claim 14 , wherein the anti-FXI antibody is raised in a first species and wherein a labeled antibody raised in a second species against antibodies from the first species is added to the solid phase and detected.  
     
     
         16 . A kit for detecting ADAMTS13/FXI complexes in a sample comprising: 
 (i) a solid phase coated with an anti-ADAMTS13 antibody; and    (ii) an anti-FXI antibody.    
     
     
         17 . The kit of  claim 16 , wherein the anti-FXI antibody is labeled.  
     
     
         18 . The kit of  claim 17 , wherein the FXI antibody is labeled with horse radish peroxidase.  
     
     
         19 . The kit of  claim 18 , further comprising a substrate for horse radish peroxidase.  
     
     
         20 . The kit of  claim 16 , further comprising one or more components selected from the group consisting of a standard sample, a positive control and a wash buffer.