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A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins

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Abstract

A surface plasmon resonance-based solution affinity assay is described for measuring the K d of binding of heparin/heparan sulfate-binding proteins with a variety of ligands. The assay involves the passage of a pre-equilibrated solution of protein and ligand over a sensor chip onto which heparin has been immobilised. Heparin sensor chips prepared by four different methods, including biotin–streptavidin affinity capture and direct covalent attachment to the chip surface, were successfully used in the assay and gave similar K d values. The assay is applicable to a wide variety of heparin/HS-binding proteins of diverse structure and function (e.g., FGF-1, FGF-2, VEGF, IL-8, MCP-2, ATIII, PF4) and to ligands of varying molecular weight and degree of sulfation (e.g., heparin, PI-88, sucrose octasulfate, naphthalene trisulfonate) and is thus well suited for the rapid screening of ligands in drug discovery applications.

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Notes

  1. Typical range of K d values is 200 nM to 200 pM. BIAapplications Handbook, version AB, 1998.

  2. Most assays are performed at flow rates of 20–40 μL/min which will further reduce the contact time.

Abbreviations

HS:

heparan sulfate

GAG:

glycosaminoglycan

SPR:

surface plasmon resonance

FGF-1:

fibroblast growth factor 1

FGF-2:

fibroblast growth factor 2

VEGF:

vascular endothelial growth factor

IL-8:

interleukin 8

MCP-2:

monocyte chemotactic protein 2

PF4:

platelet factor 4

ATIII:

antithrombin III

ADHZ:

adipic acid dihydrazide

NHS:

N-hydroxysuccinimide

EDC:

N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide

LMWH:

low molecular weight heparin

NTS:

1,3,6-naphthalenetrisulfonate

SOS:

sucrose octasulfate

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Acknowledgements

We wish to thank Mr. Rick Filonzi (Biacore AB, Australia) for his technical support, Ms. Anna Bezos and Prof. Chris Parish (John Curtin School of Medical Resarch, Australian National University) for useful discussions and Dr. A. Satoh (Ochanomizu University, Japan) for his technical advice on the immobilisation of heparin onto the CM5 sensor chip. We also thank Dr. Ian Bytheway (Progen) for useful discussions, critical reading of this manuscript, and the preparation of the figures.

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  1. Vito Ferro

    Present address: School of Physical and Chemical Sciences, Queensland University of Technology, GPO Box 2434, Brisbane, QLD, 4001, Australia

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  1. Drug Design Group, Progen Pharmaceuticals Ltd, Darra, QLD, Australia

    Siska Cochran, Cai Ping Li & Vito Ferro

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  1. Siska Cochran
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Correspondence to Vito Ferro.

Electronic supplementary material

Below is the link to the electronic supplementary material. Representative sensorgrams for each protein (FGF-1, FGF-2, VEGF, PF4, ATIII, MCP-2 and IL-8) and ligand (heparin, LMWH, PI-88, SOS and NTS) discussed in the text. Data from the negative control flow cells showing the level of non-specific binding are also provided.

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Cochran, S., Li, C.P. & Ferro, V. A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins. Glycoconj J 26, 577–587 (2009). https://doi.org/10.1007/s10719-008-9210-0

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