An enzyme-linked immunosorbent assay using biotinylated heparan sulfate to evaluate the interactions of heparin-like molecules and basic fibroblast growth factor

Analytical Biochemistry
C FoxallZ Wei

Abstract

Basic fibroblast growth factor (bFGF) is a member of the heparin-binding growth factor family that interacts with cell surface heparan sulfate (HS) proteoglycans and extracellular matrix heparin. Here we report the development of a simple and sensitive assay that used biotinylated HS or heparin to bind to bFGF coated onto 96-well microtiter plates. Bound labeled HS or heparin was reacted with enzyme-linked streptavidin and results were recorded as optical density. Increased molar excess of biotin resulted in increased incorporation of biotin and higher signal without compromising binding. Glycosaminoglycans and modified heparins were assayed for their ability to compete with biotinylated HS for binding to bFGF. Inhibition of that binding by heparin and HS but not by chondroitin sulfate A or C, dermatan sulfate, or keratan sulfate demonstrated the specificity of the glycosaminoglycan binding. Structural modifications of heparin produced various degrees of inhibition with high structural specificity. Although removal of N-sulfates or 2,3-O-sulfate groups resulted in significant loss of inhibition, removal of 6-O-sulfates had little affect on binding. Carboxyl reduction or N-acetylation following N-desulfation produced heparinoids...Continue Reading

Citations

Mar 31, 2005·Journal of Neurosurgery·Yushi UenoTakeo Fukushima
Feb 27, 2003·Proceedings of the National Academy of Sciences of the United States of America·Rahul RamanRam Sasisekharan
May 12, 2017·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Øystein Arlov, Gudmund Skjåk-Bræk

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