Methods and kits for measuring von Willebrand factor
Inventors
Assignees
Versiti Blood Research Institute Foundation Inc • Medical College of Wisconsin
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10495651-B2
Publication Date
2019-12-03
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 invention provides an agonistless method for measuring von Willebrand factor (VWF) by using a surface comprising immobilized platelet glycoprotein Ibα (GPIbα) or a functional fragment thereof. The immobilized GPIbα or functional fragment comprises at least two mutations selected from G233V, D235Y, and M239V relative to SEQ ID NO:2, with one of the mutations being D235Y. The surface is contacted with a sample having or suspected of having VWF without using a platelet aggregation agonist.
After contacting, the method detects a complex of VWF and GPIbα. Detection is performed by detecting a complex of VWF and GPIbα and, in certain formats, by using a labeled anti-VWF antibody. The approach avoids use of platelet agglutination agonists such as ristocetin and botrosctetin-free conditions, while still enabling binding between VWF and the mutated GPIbα.
The invention further describes assay implementations and formats using recombinant gain-of-function platelet glycoprotein Ibα with immobilized mutated GPIbα on a cell or solid surface. It includes solid-phase surfaces and assay formats such as FACS/FC and ELISA, and it describes test performance across VWD patient sample groups including type 2B, type 2M, platelet-type/pseudo VWD, and type 3, with improved or altered correlation with VWF function relative to VWF:RCo limitations. The document also supports alternative surface implementations using host cell surfaces that do not naturally express GPIbα, optionally including additional glycoproteins such as GPIbβ and GP-IX with specified SEQ ID identifiers.
Claims Coverage
The document includes one independent claim directed to an agonistless VWF measurement method using an immobilized, mutation-containing platelet glycoprotein Ibα surface and detecting a VWF–GPIbα complex. The claim set further constrains the independent claim through multiple dependent claim inventive features, including specific mutation combinations, particular immobilization materials, host cell surface implementations, optional co-expression of additional glycoproteins, and labeled anti-VWF antibody detection.
Agonistless immobilized mutated GPIbα surface for VWF measurement
A method of measuring VWF without using a platelet agglutination agonist by providing a surface comprising immobilized platelet glycoprotein Ibα (GPIbα) or a functional fragment thereof, 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.
Specific D235Y-containing mutation set
The method uses at least two mutations selected from G233V, D235Y and M239V relative to SEQ ID NO:2, wherein one of the mutations is D235Y, including the full set of three mutations in one refinement: D235Y, G233V, and M239V.
Solid-phase immobilization material selection
The method is performed on a solid-phase surface selected from agarose, glass, latex, or plastic.
Host cell surface implementation lacking native GPIbα expression
The method is carried out on a host cell surface where the host cell does not naturally express GPIbα.
Optional co-implementation of GPIbβ and GP-IX on host cell surface
In a method variant, the host cell surface includes glycoprotein Ibβ (GPIbβ) and optionally glycoprotein IX (GP-IX), where GPIbβ has SEQ ID NO:4 and GP-IX has SEQ ID NO:8.
Labeled anti-VWF antibody detection of VWF–GPIbα complex
Detecting a complex of VWF and GPIbα using a labeled anti-VWF antibody.
Across the claims, the inventive coverage centers on agonistless VWF measurement by contacting a VWF-containing sample with an immobilized GPIbα (or functional fragment) bearing at least two specified mutations including D235Y, followed by detection of a VWF–GPIbα complex. Dependent refinements further specify mutation combinations, immobilization format/materials, host-cell surface constraints, optional co-expression of GPIbβ/GP-IX with defined SEQ IDs, and labeled anti-VWF antibody detection.
Stated Advantages
Measures VWF without using a platelet agglutination agonist.
Enables detection of a complex of VWF and GPIbα under agonistless conditions.
Documented Applications
VWF functional measurement using an agonistless approach across VWD patient sample groups including type 2B, type 2M, platelet-type/pseudo VWD, and type 3, with assay readouts including ELISA and FACS.
Interested in licensing this patent?