Methods and kits for measuring von willebrand factor
Methods and kits for measuring levels of von Willebrand factor function in a sample without using a platelet aggregation agonist, such as ristocetin, comprising recombinant glycoprotein Ibα having at least two of a G233V, D235Y and M239V mutations and an agent to detect a complex between the recombinant glycoprotein Ibα and von Willebrand factor.
1. An isolated GPIbα polypeptide or functional fragment thereof, comprising an amino acid sequence that has at least 90% sequence identity to residues 1-290 of SEQ ID NO:11 and that includes at least two mutations relative to residues 1-290 of SEQ ID NO:11, wherein one of the mutations is D235Y;
wherein the isolated GPIbα polypeptide or fragment is capable of forming a complex with von Willebrand factor (VFW).
2. The isolated GPIbα polypeptide or functional fragment of claim 1 , wherein the second mutation in the amino acid sequence having at least 90% sequence identity to residues 1-290 of SEQ ID NO:11 is selected from the group consisting of G233V and M239V.
3. The isolated GPIbα polypeptide or functional fragment of claim 2 , wherein the amino acid sequence having at least 90% sequence identity to residues 1-290 of SEQ ID NO:11 includes the three mutations D235Y/G233V/M239V.
4. The isolated GPIbα polypeptide or functional fragment of claim 1 , wherein the amino acid sequence having at least 90% sequence identity to residues 1-290 of SEQ ID NO:11 includes a mutation to prevent dimerization with a second GPIbα polypeptide.
5. The isolated GPIbα polypeptide or functional fragment of claim 1 , having a C65A mutation.
6. The isolated GPIbα polypeptide or functional fragment of claim 1 , wherein the amino acid sequence having at least 90% sequence identity to residues 1-290 of SEQ ID NO:11 lacks a transmembrane region.
7. The isolated GPIbα polypeptide or functional fragment of claim 1 , wherein the polypeptide or functional fragment is coated to a solid-phase surface.
8. The isolated GPIbα polypeptide or functional fragment of claim 7 , wherein the amino acid sequence having at least 90% sequence identity to residues 1-290 of SEQ ID NO:11 has one or both of a G233V or an M239V mutation.
9. The isolated GPIbα polypeptide or functional fragment of claim 8 , wherein the polypeptide or functional fragment is further modified to prevent dimerization with a second GPIbα polypeptide, lacks a transmembrane region or both.
10. The isolated GPIbα polypeptide or functional fragment of claim 8 , wherein the polypeptide is coated to a solid-phase surface using an anti-GPIbα monoclonal antibody.
11. The isolated GPIbα polypeptide or functional fragment of claim 10 , wherein the amino acid sequence having at least 90% sequence identity to residues 1-290 of SEQ ID NO:11 has a C65A mutation.
12. The isolated GPIbα polypeptide or functional fragment of claim 10 , wherein the polypeptide lacks a transmembrane region.
13. An ELISA assay kit for measuring vonWillebrand activity, wherein the kit contains the GPIbα polypeptide or functional fragment of claim 1 .
14. The ELISA assay kit of claim 13 , wherein the polypeptide or functional fragment is coated to a solid-phase surface.
15. A method for diagnosing vonWillebrand disease (VWD) comprising:
contacting a biological sample from an individual suspected of having VWD with the polypeptide or functional fragment of claim 1 ; and
detecting a complex between the polypeptide or functional fragment and vonWillebrand Factor (VWF).
16. The method of claim 15 , wherein the method is a vonWillebrand Factor activity assay and the method is performed in the presence of a platelet aggregation agonist.
17. The method of claim 16 , wherein the method is a vonWillebrand Factor activity assay and the method is performed in the presence of from about 0 mg/ml to about 1.2 mg/ml of Ristocetin.
18. An ELISA assay kit for measuring vonWillebrand activity, wherein the kit contains the GPIbα polypeptide or functional fragment of claim 2 .
19. The ELISA assay kit of claim 18 , wherein the wherein the polypeptide or functional fragment is coated to a solid-phase surface.
20. An ELISA assay kit for measuring vonWillebrand activity, wherein the kit contains the GPIbα polypeptide or functional fragment of claim 3 .
21. The ELISA assay kit of claim 20 , wherein the wherein the polypeptide or functional fragment is coated to a solid-phase surface.
22. A method for diagnosing vonWillebrand disease comprising:
contacting a biological sample from an individual suspected of having VWD with the polypeptide or functional fragment of claim 2 ; and
detecting a complex between the polypeptide or functional fragment and vonWillebrand Factor (VWF).
23. The method of claim 22 , wherein the method is a vonWillebrand Factor activity assay and the method is performed in the presence of a platelet aggregation agonist.
24. The method of claim 22 , wherein the method is a vonWillebrand Factor activity assay and the method is performed in the presence of from about 0 mg/ml to about 1.2 mg/ml of Ristocetin.
25. A method for diagnosing vonWillebrand disease, wherein the method uses the polypeptide or functional fragment of claim 3 .
26. The method of claim 25 , wherein the method is a vonWillebrand Factor activity assay and the method is performed in the presence of a platelet aggregation agonist.
27. The method of claim 25 , wherein the method is a vonWillebrand Factor activity assay and the method is performed in the presence of from about 0 mg/ml to about 1.2 mg/ml of Ristocetin.