Methods for detecting TAFIa or TAFIai
The invention relates to a diagnostic assay for selectively measuring levels of the 35kD form of thrombin-activatable fibrinolysis inhibitor (TAFIa or TAFIai), or a derivative or variant thereof, but not the TAFI proenzyme (TAFI) or the N-terminal activation peptide of TAFI.
1. A method for detecting active thrombin activatable fibrinolytic inhibitor (TAFIa) or inactive thrombin activatable fibrinolytic inhibitor (TAFIai), but not a 60 kD proenzyme form of thrombin activatable fibrinolytic inhibitor (TAFI) or a N-terminal activation peptide of TAFI, in a sample, said method comprising:
(i) contacting said sample with a potato carboxypeptidase inhibitor (PTCI) under conditions that permit binding of PTCI to TAFIa or TAFIai; and
(ii) detecting the presence of a TAFIa/PTCI complex or a TAFIai/PTCI complex,
wherein the TAFIa, or TAFIai or PTCI is immobilized on a solid support.
2. The method of claim 1 , wherein potato carboxypeptidase inhibitor (PTCI) is immobilized onto a solid support.
3. The method of claim 2 , wherein the solid support is a microtiter plate.
4. The method of claim 2 , wherein the solid support is a bead or microparticle.
5. The method of claim 1 , wherein said detecting step further comprises contacting said sample with an agent which binds to the TAFIa or TAFIai region of the TAFIa/PTCI complex or the TAFIai/PTCI complex.
6. The method of claim 5 , wherein said agent is an anti-TAFIa or anti-TAFIai antibody conjugated to horseradish peroxidase (HRP).
7. The method of claim 5 , wherein said agent is an anti-TAFIa or anti-TAFIai monoclonal antibody.
8. The method of claim 7 , wherein said detecting step comprises contacting said sample with an agent that binds to the anti-TAFIa or anti-TAFIai monoclonal antibody, said agent being conjugated to HRP.
9. The method of claim 8 , wherein said agent is donkey anti-mouse-IgG-HRP.
10. The method of claim 6 or 9 , wherein said detecting step comprises contacting said sample with a substrate of horseradish peroxidase (HRP).
11. The method of claim 10 , wherein said horseradish peroxidase (HRP) substrate is 3,3′,5,5′-tetramethylbenzidine (TMB).
12. The method of claim 1 , wherein the sample is a biological fluid from a patient.
13. The method of claim 12 , wherein the biological fluid is plasma.
14. The method of claim 12 , wherein the biological fluid is blood, urine, serum, semen or cerebral or spinal fluid.
15. The method of claim 12 , wherein the patient has a thrombotic disorder.
16. A method of claim 15 , wherein the thrombotic disorder is heart attack, stroke, thromboembolic disease, acute myocardial infarction (AMI), deep vein thrombosis, acute ischemic stroke, massive pulmonary embolism, disseminated intravascular coagulation (DIC), anti-phospholipid syndrome, familial thrombophilia, thrombotic thrombocytopenic purpura sepsis, arthritis, fulminant hepatitis or thrombosis.
17. The method of claim 12 , wherein the patient has a hemorrhagic disorder.
18. The method of claim 17 , wherein the hemorrhagic disorder is hemophilia A, hemophilia B, autoimmune haemolytic anaemia, a collagen disease, von Willebrand disease (VWD), Henoch-Schonlein purpura, acute-generalized widespread bleeding, primary hyperfibrinolysis, hepatosplenic schistosomiasis, or hemostasis.
19. The method of claim 12 , wherein the patient is undergoing anti-coagulant therapy.
20. The method of claim 19 , wherein the anti-coagulant therapy is administration of tissue plasminogen activator (t-PA), or urokinase plasminogen activator(u-PA).
21. The method of claim 19 , wherein said anti-coagulant therapy is administration of aspirin.
22. The method of claim 19 , wherein said anti-coagulant therapy is administration of streptokinase, staphylokinase, plasminogen, heparin or low molecular weight heparin.
23. The method of claim 12 , wherein the patient is undergoing procoagulant therapy.
24. The method of claim 23 , wherein the procoagulant therapy is administration of amino caproic acid, Factor VII, Factor VIII, Factor IX, Factor VIIa, Factor IXa, activated protein C, thrombin, fibrinogen or cryofractionalized plasma.
25. The method of claim 12 , wherein the patient is susceptible to having a thrombotic disorder.
26. The method of claim 25 , wherein the thrombotic disorder is heart attack, stroke, thromboembolic disease, acute myocardial infarction (AMI), deep vein thrombosis, acute ischemic stroke, massive pulmonary embolism, disseminated intravascular coagulation (DIC), anti-phospholipid syndrome, familial thrombophilia, thrombotic thrombocytopenic purpura sepsis, arthritis, fulminant hepatitis or thrombosis.
27. The method of claim 12 , wherein the patient is susceptible to having a hemorrhagic disorder.
28. The method of claim 27 , wherein the hemorrhagic disorder is hemophilia A, hemophilia B, autoimmune haemolytic anaemia, collagen diseases, von Willebrand disease (VWD), Henoch-Schonlein purpura, acute-generalized widespread bleeding, primary hyperfibrinolysis, hepatosplenic schistosomiasis, or hemostasis.
29. The method of claim 1 , wherein the lower limit of detection of TAFIa and TAFIai in the solution is about 0.1 μg/mL.
30. The method of claim 1 , wherein the lower limit of detection of TAFIa and TAFIai in the solution is about 0.02 μg/mL.
31. The method of claim 1 , wherein the lower limit of detection of TAFIa and TAFIai in the solution is about 0.002 μg/mL.
32. The method of claim 1 , wherein the lower limit of detection of TAFIa and TAFIai in the solution is about 0.001 μg/mL.