Analytical characterization and reference interval of an enzyme-linked immunosorbent assay for active von Willebrand factor

Lisa N. van der Vorm; Li Li; Dana Huskens; Walid Chayoua; Hilde Kelchtermans; Philip G. de Groot; Mark Roest; Jasper A. Remijn; Bas de Laat

doi:10.1371/journal.pone.0211961

Analytical characterization and reference interval of an enzyme-linked immunosorbent assay for active von Willebrand factor

Lisa N. van der Vorm^*, Li Li, Dana Huskens, Walid Chayoua, Hilde Kelchtermans, Philip G. de Groot, Mark Roest, Jasper A. Remijn, Bas de Laat

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Background

Interaction of von Willebrand factor (VWF) with platelets requires a conformational change that exposes an epitope within the VWF Al domain, enabling platelet glycoprotein Iba binding. Quantification of this "active" conformation of VWF has been shown to provide pathophysiological insight into conditions characterized by excessive VWF-platelet interaction.

Methods

We developed an immunosorbent assay based on a variable heavy chain antibody fragment against the VWF Al domain as a capture antibody. Assay performance in terms of specificity (binding to active R1306W- and sheared VWF), precision, accuracy, linearity, limits of detection and stability were determined. Active VWF, VWF antigen, VWF ristocetin cofactor activity, VWF:GP1bM and VWF propeptide were measured in citrated plasma and platelet-VWF binding in whole blood from 120 healthy individuals to establish a reference interval for active VWF and to assess associations with other VWF parameters.

Results

Intra- and inter-assay CVs were between 2.4-7.2% and 4.1-9.4%, depending on the level. Mean recovery of spiked recombinant R1306W VWF was 103 +/- 3%. The assay was linear in the range of 90.1-424.5% and had a limit of quantification of 101%. The reference interval for active VWF was 91.6-154.8% of NPP. Significant, positive correlations between active VWF and all other VWF parameters were found, with the strongest correlation with VWF: GP1bM binding.

Conclusions

We developed and validated an immunosorbent assay for the accurate detection of active VWF levels in plasma. The assay fulfilled all analytical criteria in this study and a reference interval was established, allowing its use to quantify active VWF in pathological conditions for future research.

Original language	English
Article number	0211961
Number of pages	16
Journal	PLOS ONE
Volume	14
Issue number	2
DOIs	https://doi.org/10.1371/journal.pone.0211961
Publication status	Published - 13 Feb 2019

Keywords

PLATELET-ADHESION
FACTOR-VIII
VONWILLEBRAND-FACTOR
FACTOR LEVEL
BLOOD-GROUP
DISEASE
DIAGNOSIS
ADAMTS13
AGE
ASSOCIATION

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Cite this

@article{bf449ea6c7554494ad6e944d0c139bd4,

title = "Analytical characterization and reference interval of an enzyme-linked immunosorbent assay for active von Willebrand factor",

abstract = "BackgroundInteraction of von Willebrand factor (VWF) with platelets requires a conformational change that exposes an epitope within the VWF Al domain, enabling platelet glycoprotein Iba binding. Quantification of this {"}active{"} conformation of VWF has been shown to provide pathophysiological insight into conditions characterized by excessive VWF-platelet interaction.MethodsWe developed an immunosorbent assay based on a variable heavy chain antibody fragment against the VWF Al domain as a capture antibody. Assay performance in terms of specificity (binding to active R1306W- and sheared VWF), precision, accuracy, linearity, limits of detection and stability were determined. Active VWF, VWF antigen, VWF ristocetin cofactor activity, VWF:GP1bM and VWF propeptide were measured in citrated plasma and platelet-VWF binding in whole blood from 120 healthy individuals to establish a reference interval for active VWF and to assess associations with other VWF parameters.ResultsIntra- and inter-assay CVs were between 2.4-7.2% and 4.1-9.4%, depending on the level. Mean recovery of spiked recombinant R1306W VWF was 103 +/- 3%. The assay was linear in the range of 90.1-424.5% and had a limit of quantification of 101%. The reference interval for active VWF was 91.6-154.8% of NPP. Significant, positive correlations between active VWF and all other VWF parameters were found, with the strongest correlation with VWF: GP1bM binding.ConclusionsWe developed and validated an immunosorbent assay for the accurate detection of active VWF levels in plasma. The assay fulfilled all analytical criteria in this study and a reference interval was established, allowing its use to quantify active VWF in pathological conditions for future research.",

keywords = "PLATELET-ADHESION, FACTOR-VIII, VONWILLEBRAND-FACTOR, FACTOR LEVEL, BLOOD-GROUP, DISEASE, DIAGNOSIS, ADAMTS13, AGE, ASSOCIATION",

author = "{van der Vorm}, {Lisa N.} and Li Li and Dana Huskens and Walid Chayoua and Hilde Kelchtermans and {de Groot}, {Philip G.} and Mark Roest and Remijn, {Jasper A.} and {de Laat}, Bas",

note = "Funding Information: L.L. has been awarded a scholarship (File no. 201706230245) under the State Scholarship Fund to pursue her study in the Netherlands as a PhD student by the China Scholarship Council (CSC, https://www.cscscholarship.org/). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would like to thank V. Verschuur, G. Oelemans-Prins, W. Gerritsen, M. Koldenhof-Visser, I. Bakker-Wolbers and B. Wildeboer (Gelre Hospitals Apeldoorn) for assistance with the VWF:Ag and VWF:RCo measurements and M. van Wijnen, M. Siderius and B. Blankenspoor (Meander Medical Center Amersfoort) for their help with the VWF:GP1bM measurements. We are grateful to our colleagues at Synapse Research Institute Maastricht for recruiting donors and for blood drawing and processing. Publisher Copyright: {\textcopyright} 2019 van der Vorm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.",

year = "2019",

month = feb,

day = "13",

doi = "10.1371/journal.pone.0211961",

language = "English",

volume = "14",

journal = "PLOS ONE",

issn = "1932-6203",

publisher = "Public Library of Science",

number = "2",

}

TY - JOUR

T1 - Analytical characterization and reference interval of an enzyme-linked immunosorbent assay for active von Willebrand factor

AU - van der Vorm, Lisa N.

AU - Li, Li

AU - Huskens, Dana

AU - Chayoua, Walid

AU - Kelchtermans, Hilde

AU - de Groot, Philip G.

AU - Roest, Mark

AU - Remijn, Jasper A.

AU - de Laat, Bas

N1 - Funding Information: L.L. has been awarded a scholarship (File no. 201706230245) under the State Scholarship Fund to pursue her study in the Netherlands as a PhD student by the China Scholarship Council (CSC, https://www.cscscholarship.org/). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would like to thank V. Verschuur, G. Oelemans-Prins, W. Gerritsen, M. Koldenhof-Visser, I. Bakker-Wolbers and B. Wildeboer (Gelre Hospitals Apeldoorn) for assistance with the VWF:Ag and VWF:RCo measurements and M. van Wijnen, M. Siderius and B. Blankenspoor (Meander Medical Center Amersfoort) for their help with the VWF:GP1bM measurements. We are grateful to our colleagues at Synapse Research Institute Maastricht for recruiting donors and for blood drawing and processing. Publisher Copyright: © 2019 van der Vorm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

PY - 2019/2/13

Y1 - 2019/2/13

N2 - BackgroundInteraction of von Willebrand factor (VWF) with platelets requires a conformational change that exposes an epitope within the VWF Al domain, enabling platelet glycoprotein Iba binding. Quantification of this "active" conformation of VWF has been shown to provide pathophysiological insight into conditions characterized by excessive VWF-platelet interaction.MethodsWe developed an immunosorbent assay based on a variable heavy chain antibody fragment against the VWF Al domain as a capture antibody. Assay performance in terms of specificity (binding to active R1306W- and sheared VWF), precision, accuracy, linearity, limits of detection and stability were determined. Active VWF, VWF antigen, VWF ristocetin cofactor activity, VWF:GP1bM and VWF propeptide were measured in citrated plasma and platelet-VWF binding in whole blood from 120 healthy individuals to establish a reference interval for active VWF and to assess associations with other VWF parameters.ResultsIntra- and inter-assay CVs were between 2.4-7.2% and 4.1-9.4%, depending on the level. Mean recovery of spiked recombinant R1306W VWF was 103 +/- 3%. The assay was linear in the range of 90.1-424.5% and had a limit of quantification of 101%. The reference interval for active VWF was 91.6-154.8% of NPP. Significant, positive correlations between active VWF and all other VWF parameters were found, with the strongest correlation with VWF: GP1bM binding.ConclusionsWe developed and validated an immunosorbent assay for the accurate detection of active VWF levels in plasma. The assay fulfilled all analytical criteria in this study and a reference interval was established, allowing its use to quantify active VWF in pathological conditions for future research.

AB - BackgroundInteraction of von Willebrand factor (VWF) with platelets requires a conformational change that exposes an epitope within the VWF Al domain, enabling platelet glycoprotein Iba binding. Quantification of this "active" conformation of VWF has been shown to provide pathophysiological insight into conditions characterized by excessive VWF-platelet interaction.MethodsWe developed an immunosorbent assay based on a variable heavy chain antibody fragment against the VWF Al domain as a capture antibody. Assay performance in terms of specificity (binding to active R1306W- and sheared VWF), precision, accuracy, linearity, limits of detection and stability were determined. Active VWF, VWF antigen, VWF ristocetin cofactor activity, VWF:GP1bM and VWF propeptide were measured in citrated plasma and platelet-VWF binding in whole blood from 120 healthy individuals to establish a reference interval for active VWF and to assess associations with other VWF parameters.ResultsIntra- and inter-assay CVs were between 2.4-7.2% and 4.1-9.4%, depending on the level. Mean recovery of spiked recombinant R1306W VWF was 103 +/- 3%. The assay was linear in the range of 90.1-424.5% and had a limit of quantification of 101%. The reference interval for active VWF was 91.6-154.8% of NPP. Significant, positive correlations between active VWF and all other VWF parameters were found, with the strongest correlation with VWF: GP1bM binding.ConclusionsWe developed and validated an immunosorbent assay for the accurate detection of active VWF levels in plasma. The assay fulfilled all analytical criteria in this study and a reference interval was established, allowing its use to quantify active VWF in pathological conditions for future research.

KW - PLATELET-ADHESION

KW - FACTOR-VIII

KW - VONWILLEBRAND-FACTOR

KW - FACTOR LEVEL

KW - BLOOD-GROUP

KW - DISEASE

KW - DIAGNOSIS

KW - ADAMTS13

KW - AGE

KW - ASSOCIATION

U2 - 10.1371/journal.pone.0211961

DO - 10.1371/journal.pone.0211961

M3 - Article

C2 - 30759116

SN - 1932-6203

VL - 14

JO - PLOS ONE

JF - PLOS ONE

IS - 2

M1 - 0211961

ER -