Methods and Kits for Determining von Willebrand Factor Activity in the Absence of Ristocetin and for Determining the Activity of ADAMTS-13 Protease
AssigneeSiemens Healthcare Diagnostics Products GmbH
InventorsHarald Althaus, Tobias Obser, Juergen Patzke, Reinhard Schneppenheim
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Abstract
Described herein are method(s), kit(s), reagent(s) and the like for determining von Willebrand factor (VWF) activity in a sample in the absence of ristocetin.
Claims45
A method for determining von Willebrand factor (VWF) activity in a sample, wherein the sample is mixed with isolated glycoprotein lb heavy chain (GPlbα) protein to give an assay mix, comprising the steps of:
- a) providing an isolated GPlbα protein associated with a particulate solid phase, wherein the isolated GPlbα protein comprises an amino acid sequence which, when compared to the wild-type sequence of human GPlbα protein (SEQ ID NO:
- 1), includes at least the amino acid residues 1-268 of SEQ ID NO:
- 1 except that amino acid substitutions are found in positions 233 and 239;
- b) adding to the assay mix neither ristocetin nor botrocetin;
- and
- c) determining the VWF activity in the assay mix.
A method for determining von Willebrand factor (VWF) activity in a sample, where the sample is mixed with isolated glycoprotein lb heavy chain (GPlbα) protein to give an assay mix, comprising the steps of:
- a) providing a sample;
- b) providing an isolated GPlbα protein associated with a particulate solid phase, wherein the isolated GPlbα protein comprises SEQ ID NO:
- 2;
- c) mixing the sample with the isolated GPlbα protein to provide an assay mix, wherein neither ristocetin nor botrocetin are present in the assay mix;
- and
- d) determining the VWF activity in the assay mix.
A method for determining the von Willebrand factor (VWF)-cleaving activity of a disintegrin metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) protease in a sample, comprising the steps of:
- a) mixing a sample which is suspected of containing the ADAMTS-13 protease with isolated VWF as a substrate to provide a reaction mix;
- b) incubating the reaction mix;
- and
- c) determining residual VWF activity in the reaction mix, wherein the VWF substrate is a high molecular weight, multimeric VWF which includes VWF monomers having at least one amino acid sequence variation resulting in said VWF substrate being broken down by ADAMTS-13 in an accelerated manner compared to a VWF substrate that is high molecular weight, multimeric VWF composed of wild-type VWF monomers.
A method for determining VWF activity in a competitive assay, the method comprising the steps of:
- a) providing a sample which is suspected of containing VWF;
- b) providing particle-associated anti-GPlbα antibody in a reaction mix;
- c) adding particle-associated VWF to the reaction mix;
- d) adding the sample to the reaction mix;
- e) adding GPlbα protein comprising an amino acid sequence which, when compared to the wild-type sequence of human GPlbα protein (SEQ ID NO:1), includes at least amino acid residues 1-268 of SEQ ID NO:1 except that amino acid substitutions are found in positions 233 and 239 to the reaction mix and mixing same in the absence of ristocetin and botrocetin, wherein agglutination occurs in the absence of VWF;
- and
- f) determining that VWF is present in the sample if the agglutination reaction is inhibited.
Dependencies are inferred from the claim text.