Methods for testing effectiveness of a von Willebrand factor used in treating von Willebrand disease (VWD)
The invention generally relates to methods of testing the effectiveness of a von Willebrand factor (VWF) in treating von Willebrand disease (VWD) in a subject comprising measuring VWF cleavage fragments in a blood sample from the subject before and after treatment. In particular, the invention relates to methods of measuring VWF cleavage fragments, wherein an increase in VWF cleavage fragments after the treatment indicates that endogenous a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) in the subject is cleaving the VWF and wherein a decrease or absence of VWF cleavage fragments after the treatment indicates that endogenous ADAMTS13 in the subject is not cleaving the VWF.
1. A method for testing effectiveness of a von Willebrand Factor (VWF) used in treating von Willebrand disease (VWD) in a subject comprising measuring VWF cleavage fragments in a blood sample from the subject before and after treatment, wherein an increase in VWF cleavage fragments after the treatment indicates that endogenous disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13 ) in the subject is cleaving the VWF and wherein a decrease or absence of VWF cleavage fragments after the treatment indicates that endogenous ADAMTS13 in the subject is not cleaving the VWF;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; and the measuring of VWF cleavage fragment level comprises performing Western blot analysis with a VWF antibody to visualize VWF cleavage fragments via enhanced chemiluminescence (ECL); 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; and the measuring of VWF cleavage fragment level comprises performing Western blot analysis with a VWF antibody to visualize VWF cleavage fragments via enhanced chemiluminescence (ECL).
2. The method of claim 1 , wherein the Western blot analysis is carried out under non-reducing conditions.
3. The method of claim 1 , wherein VWF fragments are visualized through use of a VWF antibody conjugated to a marker.
4. The method of claim 3 , wherein the marker is alkaline phosphatase (ALP) or horseradish peroxidase (HRP).
5. The method of claim 1 wherein the VWF antibody is monoclonal or polyclonal.
6. 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.
7. The method of claim 1 , wherein the blood sample is plasma or serum.
8. The method of claim 7 , wherein the blood sample is plasma.
9. The method of claim 1 , wherein the subject is a mammal.
10. The method of claim 9 , wherein the mammalian subject is human, rabbit, monkey, dog, rat, mouse, or pig.
11. The method of claim 10 , wherein the subject is human, rabbit, monkey, or dog.
12. The method of claim 11 , wherein the subject is human.
13. The method of claim 1 , wherein the VWF fragment is a 176 kDa VWF fragment.
14. The method of claim 1 , wherein the VWF cleavage fragments are generated by the activity of the ADAMTS13 and carried out under shear stress.
15. The method of claim 14 , wherein the shear stress comprises a shear rate of 100 to 10,000 s-1.
16. The method of claim 15 , wherein the shear rate is 1,000 s-1 to 8,000 s-1.
17. The method of claim 15 , wherein the shear rate is 6,000 s-1.
18. The method of claim 14 , wherein the shear stress comprises a shear rate of 100 to 10,000 s-1 at a temperature of 20 ° C. to 40 ° C. for a period of time.
19. The method of claim 18 , wherein the temperature is 30 ° C. to 40 ° C.
20. The method of claim 19 , wherein the temperature is 37 ° C.
21. The method of claim 18 , wherein the period of time ranges from 30 seconds to 1 hour.
22. The method of claim 21 , wherein the time ranges from 15 minutes to 30 minutes.
23. The method of claim 22 , wherein the time is 30 minutes or is 15 minutes.