Methods of measuring adamts 13 activity
The invention generally relates to methods of measuring cleaved von Willebrand factor (VWF) fragments. More specifically, the invention relates to methods of measuring the ability of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) to cleave VWF in vivo. The invention also relates to methods of using various animal models which demonstrate ADAMTS13 activity similar to that of a human. The invention further relates to methods of measuring the cleavage products of rVWF in mammals, particularly in humans and in human plasma.
1. A method for measuring a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity in a blood sample from a subject comprising the steps of:
(a) measuring von Willebrand factor (VWF) cleavage fragments in the blood sample;
(b) comparing the VWF cleavage fragments to a reference curve of different amounts of completely degraded VWF; and
(c) quantifying an amount of ADAMTS13 activity based on the reference curve, wherein an amount of VWF cleavage fragments correlates with the amount of ADAMTS13 activity.
2. The method of claim 1 wherein the measuring of VWF cleavage fragment level comprises performing Western blot analysis with a VWF antibody to visualize VWF cleavage fragments.
3. The method of claim 2 wherein the Western blot analysis is carried out under non-reducing conditions.
4. The method of claim 2 wherein VWF fragments are visualized through use of a VWF antibody conjugated to a marker.
5. The method of claim 4 wherein the marker is alkaline phosphatase (ALP) or horseradish peroxidase (HRP).
6. The method of claim 4 wherein the marker is detected with enhanced chemiluminescence (ECL).
7. The method of claim 2 wherein the VWF antibody is monoclonal or polyclonal.
8. The method of claim 1 wherein VWF cleavage fragments are detected at a sensitivity level of about 0.025 to about 0.05 Ag U/mL VWF.
9. The method of claim 1 wherein the blood sample is plasma or serum.
10. The method of claim 9 wherein the blood sample is plasma.
11. The method of claim 1 wherein the subject is a mammal.
12. The method of claim 11 wherein the mammalian subject is human, rabbit, monkey, dog, rat, mouse, or pig.
13. The method of claim 12 wherein the subject is human, rabbit, monkey, or dog.
14. The method of claim 13 wherein the subject is human.
15. The method of claim 1 wherein a change in VWF cleavage fragment level is detected by measuring the level of one or more of a 140 kDa VWF fragment or a 176 kDa VWF fragment.
16. The method of claim 15 wherein the VWF fragment is a 176 kDa VWF fragment.
17. The method of claim 1 , wherein the VWF cleavage fragments are generated by the activity of the ADAMTS13 and carried out under shear stress.
18. The method of claim 17 , wherein the shear stress comprises a shear rate of 100 to 10,000 s−1.
19. The method of claim 18 , wherein the shear rate is 1,000 s−1 to 8,000 s−1.
20. The method of claim 18 , wherein the shear rate is 6,000 s−1.
21. A method of assessing a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity in a subject comprising measuring total VWF and VWF cleavage fragments; comparing total VWF and VWF cleavage fragment level in a blood sample of the subject to a reference curve of increasingly degraded to completely degraded VWF; and assessing ADAMTS13 activity, wherein the VWF cleavage fragment level in the blood sample correlates to the ADAMTS13 activity deduced from the reference curve.
22. The method of claim 21 , wherein the VWF cleavage fragments are generated by the activity of the ADAMTS13 and carried out under shear stress.
23. The method of claim 22 , wherein the shear stress comprises a shear rate of 100 to 10,000 s−1.
24. The method of claim 23 , wherein the shear rate is 1,000 s−1 to 8,000 s−1.
25. The method of claim 23 , wherein the shear rate is 6,000 s−1.
26. The method of claim 21 wherein the blood sample is plasma or serum.
27. The method of claim 26 wherein the blood sample is plasma.
28. The method of claim 21 wherein the subject is a mammal.
29. The method of claim 28 wherein the mammalian subject is human, rabbit, monkey, dog, rat, mouse, or pig.
30. The method of claim 29 wherein the subject is human.
31. The method of claim 21 wherein a change in VWF cleavage fragment level is detected by measuring the level of one or more of a 140 kDa VWF fragment or a 176 kDa VWF fragment.
32. The method of claim 31 wherein the VWF fragment is a 176 kDa VWF fragment.