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-8865415-B2
Publication Date
2014-10-21
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 assays for measuring von Willebrand factor (VWF) function by detecting VWF binding to recombinant platelet glycoprotein Ibα (GPIbα) bearing gain-of-function mutation sets that include G233V, D235Y, and M239V, including combinations with D235Y. The method measures VWF without using a platelet agglutination agonist by contacting a patient sample with a surface comprising immobilized GPIbα or a functional fragment and detecting a complex of VWF and GPIbα.
The disclosed assay uses immobilized GPIbα on solid-phase surfaces for VWF binding detection, including ELISA formats using immobilized GPIbα on surfaces such as agarose, glass, latex, or plastic. The disclosure also describes assay formats using engineered host-cell surfaces for detection by FACS, where the host cell does not natively express GPIbα and may optionally co-express GPIbβ and GP-IX.
The disclosure presents results indicating that engineered mutant GPIbα supports ristocetin-independent binding, including spontaneous VWF binding in the absence of ristocetin for mutant GPIbα and ristocetin-independent binding for double mutants. The assay readouts are described as distinguishing VWD types using VWF:IbCo readouts while avoiding pitfalls associated with ristocetin-dependent readouts, and patient-sample comparisons are described as yielding measurements comparable to clinical laboratory methods and improved correlation with VWF function.
Claims Coverage
The independent claim covers a ristocetin/platelet agglutination-agonist-free method for measuring VWF by detecting complexes between patient VWF and immobilized, mutation-defined platelet glycoprotein Ibα (GPIbα). Dependent claims further refine detection reagents, surface formats (solid-phase or host-cell surfaces), and additional glycoprotein components and specific mutation combinations.
Measuring VWF without using a platelet agglutination agonist via immobilized mutation-defined GPIbα complex detection
Providing a surface comprising immobilized platelet glycoprotein Ibα (GPIbα) or a functional fragment thereof, wherein the immobilized GPIbα or functional fragment comprises a combination of mutations selected from G233V, D235Y and M239V relative to SEQ ID NO:2, wherein one of the mutations is D235Y; contacting a patient sample having or suspected of having VWF with the surface without a platelet aggregation agonist; and detecting a complex of VWF and GPIbα.
Labeled anti-VWF antibody detection of the VWF–GPIbα complex
Detecting a complex of VWF and GPIbα using a labeled anti-VWF antibody.
Solid-phase surface immobilization for the GPIbα assay
Performing the method using a solid-phase surface selected from agarose, glass, latex, or plastic.
Host cell surface format with non-natively expressing GPIbα
Performing the method on a host cell surface where the host cell does not natively express GPIbα.
Mutation combination restriction including D235Y with G233V or M239V
Including a combination of mutations that is either D235Y/G233V or D235Y/M239V.
Optional GPIbβ and GP-IX on the host cell surface specified by sequence IDs
Including a host cell surface having glycoprotein Ibβ (GPIbβ) and optionally glycoprotein IX (GP-IX), where GPIbβ corresponds to SEQ ID NO:4 and GP-IX corresponds to SEQ ID NO:8.
Overall claim coverage centers on detecting VWF binding to immobilized mutation-defined GPIbα without using a platelet agglutination agonist, with dependent features specifying labeled anti-VWF detection, solid-phase material options, host-cell surface formats, defined mutation combinations involving D235Y, and optional inclusion of GPIbβ and optionally GP-IX on host cells.
Stated Advantages
Enables measuring VWF without using a platelet agglutination agonist.
Distinguishes VWD types using VWF:IbCo readouts.
Avoids pitfalls associated with ristocetin-dependent VWF:RCo measurement, including cases of normal binding despite low RCo/Ag.
Produces patient-sample measurements comparable to clinical laboratory methods and improved correlation with VWF function.
Documented Applications
Assaying patient samples having or suspected of having VWF to measure VWF function and distinguish von Willebrand disease (VWD) types.
Using ELISA formats (immobilized GPIbα on solid-phase surfaces such as agarose, glass, latex, or plastic) to detect VWF–GPIbα complexes.
Using FACS formats on engineered host-cell surfaces not natively expressing GPIbα, optionally co-expressing GPIbβ and GP-IX, to detect VWF binding.
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