Methods and kits for measuring von Willebrand factor
Inventors
Assignees
BLOOD CENTER OF WISCONSIN • Versiti Blood Research Institute Foundation Inc • Medical College of Wisconsin • Blood Center Research Foundation Inc
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Publication Number
US-8163496-B2
Publication Date
2012-04-24
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 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.
Core Innovation
The invention provides a ristocetin/platelet-aggregation agonist-free VWF function assay format that measures a complex of VWF and platelet glycoprotein Ib (GPIb). The assay uses an immobilized recombinant gain-of-function GPIb, or a functional fragment thereof, comprising at least two mutations selected from G233V, D235Y and M239V relative to SEQ ID NO: 11, with one mutation being D235Y. Contacting of a sample having or suspected of having VWF with the immobilized mutant GPIb is performed without a platelet aggregation agonist.
The disclosed approach measures VWF without using a platelet agglutination agonist by providing a surface comprising immobilized GPIb or a functional fragment thereof and then measuring the complex of VWF and GPIb. The mutant GPIb produces VWF binding or spontaneous interaction in the absence of ristocetin, including ristocetinless and botrocetinless behavior. The immobilized mutant GPIb is presented as part of the assay surface, including embodiments where the GPIb is on a host cell surface or on a solid-phase surface.
The invention further includes kit embodiments for measuring active VWF, comprising recombinant GPIb, or a functional fragment, with at least two mutations including D235Y relative to SEQ ID NO: 11 and a reagent to detect a complex of VWF and GPIb. The document describes detection modalities including labeled anti-VWF antibodies, and also notes SPR and QELS. Patient plasma analysis is described across cohorts including normals and von Willebrand disease subtypes such as type 2B, type 2M or apparent 2M, and type 3, with comparisons to conventional VWF:RCo assay behavior.
Claims Coverage
The provided portion includes three independent claims. Each independent claim centers on measuring or detecting a VWF-GPIb complex without a platelet aggregation agonist by using an immobilized or kit-delivered recombinant GPIb comprising specified gain-of-function mutations, including D235Y, constrained to at least two mutations selected from G233V, D235Y, and M239V.
Platelet-aggregation agonist-free VWF measurement with immobilized mutant GPIb
Providing a surface comprising immobilized platelet glycoprotein Ib, or a functional fragment thereof, wherein the immobilized GPIb or functional fragment thereof comprises at least two mutations selected from G233V, D235Y and M239V relative to SEQ ID NO: 11, wherein one of the mutations is D235Y; contacting a sample having or suspected of having VWF with the surface, wherein the contacting is done without a platelet aggregation agonist; and measuring a complex of VWF and GPIb.
Platelet-aggregation agonist-free VWF measurement with constrained mutation set
Providing a surface comprising immobilized platelet glycoprotein Ib, or a functional fragment thereof, wherein the immobilized GPIb or functional fragment thereof comprises at least two mutations selected from G233V, D235Y and M239V relative to SEQ ID NO: 11, wherein the at least two mutations are D235Y/G233V/M239V; contacting a sample having or suspected of having VWF with the surface, wherein the contacting is done without a platelet aggregation agonist; and measuring a complex of VWF and GPIb.
Kit for measuring active VWF using recombinant mutant GPIb and detection reagent
A kit for measuring active von Willebrand factor (VWF), the kit comprising recombinant platelet glycoprotein Ib, or a functional fragment thereof, wherein the GPIb or functional fragment thereof comprises at least two mutations selected from G233V, D235Y and M239V relative to SEQ ID NO: 11, wherein one of the mutations is D235Y; and a reagent to detect a complex of VWF and GPIb.
Across the independent claims, the inventive scope is directed to detecting a VWF-GPIb complex without using a platelet aggregation agonist by employing GPIb, or a functional fragment, containing specified mutations including D235Y, with measurement performed by contacting a VWF-containing sample with an immobilized mutant GPIb surface or by using a kit comprising recombinant mutant GPIb plus a reagent to detect the resulting VWF-GPIb complex.
Stated Advantages
Measures VWF without using a platelet agglutination agonist.
Enables VWF binding or spontaneous interaction in the absence of ristocetin, including ristocetinless and botrocetinless behavior.
Documented Applications
Measuring active von Willebrand factor (VWF) in a sample having or suspected of having VWF, including analysis of patient plasma across normals and VWD subtypes such as type 2B, type 2M or apparent 2M, and type 3.
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