Methods and kits for measuring von Willebrand factor
Inventors
Assignees
Versiti Blood Research Institute Foundation Inc • Medical College of Wisconsin • Blood Center Research Foundation Inc
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Publication Number
US-9678089-B2
Publication Date
2017-06-13
Expiration Date
Abstract
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 a combination of G233V, D235Y and M239V mutations and an agent to detect a complex between the recombinant glycoprotein Ibα and von Willebrand factor.
Core Innovation
The document describes methods and kits for measuring active von Willebrand factor (VWF) without using a platelet agglutination agonist. The approach provides a solid phase surface comprising immobilized platelet glycoprotein Ibα (GPIbα) or a functional fragment thereof, and then contacts a sample suspected of having VWF with the surface without a platelet aggregation agonist. After contacting, the method detects a complex of VWF and GPIbα.
The immobilized GPIbα or functional fragment includes at least two mutations selected from G233V, D235Y and M239V relative to SEQ ID NO:2, with one of the mutations being D235Y. The document further describes kit embodiments where recombinant platelet glycoprotein Ibα (GPIbα) or a functional fragment thereof includes the same mutation requirements, together with a reagent to detect a complex of VWF and GPIbα.
Assay and detection formats described in the document include immobilized mutant GPIbα on a solid phase surface and detection of the VWF–GPIbα complex. The document also describes an approach using engineered host cells expressing mutant GPIbα, where VWF–GPIbα complex formation is detected using labeled anti-VWF antibodies or other detection approaches. The described embodiments are presented for different assay surfaces and formats, with the goal of measuring VWF function without reliance on a platelet agglutination agonist.
Claims Coverage
The partial content contains two independent claims that cover both a method and a kit for measuring active von Willebrand factor without a platelet agglutination agonist. Each independent claim centers on immobilized or provided recombinant platelet GPIbα (or a functional fragment) bearing specific gain-of-function mutations including D235Y, followed by detection of a VWF–GPIbα complex.
Ristocetinless/botrocetinless VWF measurement using immobilized mutant GPIbα
providing a solid phase surface comprising immobilized platelet glycoprotein Ibα (GPIbα) or a functional fragment thereof, wherein the solid phase surface is latex and wherein the immobilized GPIbα or functional fragment comprises at least two mutations selected from G233V, D235Y and M239V relative to SEQ ID NO:2, wherein one of the mutations is D235Y; contacting a sample having or suspected of having VWF with the surface without a platelet aggregation agonist; and detecting a complex of VWF and GPIbα.
Active VWF measuring kit with recombinant mutant GPIbα and VWF–GPIbα detection reagent
a kit comprising recombinant platelet glycoprotein Ibα (GPIbα) or a functional fragment thereof, wherein the GPIbα or functional fragment comprises at least two mutations selected from G233V, D235Y and M239V relative to SEQ ID NO:2, wherein one of the mutations is D235Y; and a reagent to detect a complex of VWF and GPIbα.
Across both independent claims, coverage is directed to VWF function/active VWF measurement by detecting a VWF–GPIbα complex using GPIbα (or a functional fragment) that bears at least two specified mutations including D235Y, with the method performed without a platelet aggregation agonist and the kit including a complex-detection reagent.
Stated Advantages
Measures VWF without using a platelet agglutination agonist.
Enables a VWF function assay that is described as ristocetinless/botrocetinless in vitro.
Is described as correlating VWF:IbCo measurements more directly with VWF function than VWF:RCo.
Works across patient plasma types including VWD subtypes and cases with atypical ristocetin-cofactor results.
Documented Applications
In vitro measurement of VWF function using FACS with engineered host cells expressing mutant GPIbα, including samples from patient plasma types and VWD subtypes, with behavior described as ristocetinless/botrocetinless binding.
ELISA evaluation using solid-phase immobilized mutant GPIbα and detection of the VWF–GPIbα complex, with comparisons described against clinical VWF:IbCo and VWF:RCo assays and performance comparisons with or without added ristocetin.
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