Assays for diagnosis of thrombophilic disease

Abstract

The present invention provides methods for diagnosing and/or monitoring thrombophilic disease in a patient that can result from the antiphospholipid antibody syndrome (aPL syndrome). The methods of the invention are premised on the inhibition of binding of an anticoagulant protein, annexin, preferably annexin-V, to phospholipids by antiphospholipid (aPL) antibodies in a patient blood sample.

I claim:  
     
         1 . A method of diagnosing and/or monitoring a thrombophilic disease in a patient comprising 
 a. incubating a phospholipid substrate with a test blood specimen from the patient in the presence of a known amount of an annexin for a time sufficient to allow the annexin to bind to the phospholipid substrate,    b. removing unbound test specimen and annexin from the phospholipid substrate,    c. measuring the amount of annexin bound to the phospholipid substrate in the presence of the test specimen, and    d. comparing the amount of annexin bound in the presence of the test specimen to the same known amount of annexin bound to a phospholipid substrate in the presence of a control blood specimen, wherein a lower amount of annexin bound to the phospholipid substrate in the presence of the test specimen compared with the control specimen indicates the presence of thrombophilic disease in the individual.    
     
     
         2 . A method of diagnosing and/or monitoring a thrombophilic disease in a patient comprising 
 a. incubating a phospholipid substrate with a test blood specimen from the patient in the presence of a known amount of an annexin for a time sufficient to allow the annexin to bind to the phospholipid substrate,    b. removing unbound test specimen and annexin from the phospholipid substrate,    c. measuring the amount of unbound annexin from step b, and    d. comparing the amount of unbound annexin from step b to an amount of unbound annexin in a sample of annexin bound to a phospholipid substrate in the presence of a control blood specimen, wherein a higher amount of unbound annexin in the presence of the test specimen compared to the amount of unbound annexin in the presence of the control specimen indicates the presence of thrombophilic disease.    
     
     
         3 . The method of    claim 1    or    2    wherein the patient has or is suspected of having the antiphospholipid syndrome.  
     
     
         4 . The method of    claim 1    or    2    wherein the annexin is annexin-V.  
     
     
         5 . The method of    claim 1    or    2    wherein the test specimen and control specimen comprise anticoagulated plasma.  
     
     
         6 . The method of    claim 1    or    2    wherein the test specimen and control specimen comprise isolated IgG.  
     
     
         7 . The method of    claim 6    wherein the incubation is carried out in the presence of a cofactor that enhances antiphospholipid antibody binding to the phospholipid substrate.  
     
     
         8 . The method of    claim 7    wherein the cofactor is β 2 -glycoprotein I.  
     
     
         9 . The method of    claim 1    or    2    wherein the phospholipid substrate is selected from among cultured trophoblasts, endothelial cells, other cells and cultured cell lines having surface anionic phospholipids, platelets, phospholipid coated silicon wafers, phospholipid coated microtiter plates, phospholipid coated beads, phospholipid suspensions and coagulation test reagents comprising phospholipids.  
     
     
         10 . The method of    claim 9    wherein the coagulation test reagent is selected from among partial thromboplastin time reagent and prothrombin reagent.  
     
     
         11 . The method of    claim 9    wherein the phospholipid substrate comprises a phospholipid selected from among phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, cardiolipin, phosphatidic acid and combinations thereof.  
     
     
         12 . The method of    claim 9    wherein the phospholipid substrate is a phospholipid coated microtiter plate or phospholipid coated beads.  
     
     
         13 . The method of    claim 9    wherein the phospholipid substrate is platelets.  
     
     
         14 . The method of    claim 9    wherein the phospholipid substrate is partial thromboplastin time reagent.  
     
     
         15 . The method of    claim 9    wherein the phospholipid substrate is prothrombin reagent.  
     
     
         16 . The method of    claim 1    or    2    wherein the annexin comprises a detectable label.  
     
     
         17 . The method of    claim 16    wherein the detectable label is selected from among biotin, a fluorochrome, a radioisotope, a chemiluminescent label and a chromophore.  
     
     
         18 . The method of    claim 17    wherein the fluorochrome is fluorescein isothiocyanate.  
     
     
         19 . The method of    claim 1    further comprising removing the bound annexin form the phospholipid substrate for measurement.  
     
     
         20 . The method of    claim 19    wherein the annexin is removed by desorbing with a calcium chelating agent.  
     
     
         21 . The method of    claim 20    wherein the calcium chelating agent is EDTA or EGTA.  
     
     
         22 . The method of    claim 1    wherein bound annexin is measured by an ELISA, streptavidin binding, fluorescence or chemiluminescence.  
     
     
         23 . The method of    claim 20    wherein desorbed annexin is measured by an ELISA, streptavidin binding, fluorescence or chemiluminescence  
     
     
         24 . A method of diagnosing and/or monitoring thrombophilic disease in a patient comprising 
 a. incubating in duplicate a phospholipid dependent coagulation test reagent with a sample of anticoagulated patient plasma,    b. calcifying the plasma in one of the duplicate sample of step a in the presence of a standardized amount of annexin and calcifying the plasma in the other sample in the absence of annexin, and    c. monitoring the time until clot formation occurs in each sample, wherein a reduced anticoagulant effect in the presence of annexin indicates thrombophilic disease in the patient.    
     
     
         25 . The method of    claim 24    wherein the phospholipid dependent coagulation test phospholipid substrate is selected from among partial thromboplastin time reagent and prothrombin reagent.  
     
     
         26 . The method of    claim 24    wherein the patient has the antiphospholipid antibody syndrome.  
     
     
         27 . The method of    claim 24    wherein the annexin is annexin-V.  
     
     
         28 . The method of    claim 24    wherein the plasma is citrated.  
     
     
         29 . A method of diagnosing and/or monitoring thrombophilic disease in a patient compising 
 a. incubating in duplicate a phospholipid dependent coagulation test reagent with a sample of anticoagulated patient plasma or isolated patient IgG,    b. removing unbound plasma or IgG from the coagulation test reagent,    c. incubating the duplicate samples of step b with a sample of control plasma,    d. calcifying the control plasma in one of the duplicate samples of step c in the presence of a standardized amount of annexin and calcifying the control plasma in the other sample in the absence of annexin, and    e. monitoring the time until clot formation occurs in each sample, wherein a reduced anticoagulant effect in the presence of annexin indicates thrombophilic disease in the patient.    
     
     
         30 . The method of    claim 25    wherein the phospholipid dependent coagulation test phospholipid substrate is selected from among partial thromboplastin time reagent and prothrombin reagent.  
     
     
         31 . The method of    claim 26    wherein the patient has the antiphospholipid antibody syndrome.  
     
     
         32 . The method of    claim 27    wherein the annexin is annexin-V.  
     
     
         33 . The method of    claim 28    wherein the plasma is citrated.  
     
     
         34 . The method of    claim 28    wherein the control plasma is pooled normal plasma.