An enzyme-linked immunosorbent assay using biotinylated heparan sulfate to evaluate the interactions of heparin-like molecules and basic fibroblast growth factor.
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 with moderate changes in binding capacity. Results from this assay are in agreement with previous data from our laboratory and reports from other researchers with respect to the specificity of glycosaminoglycan binding to bFGF and the role of 2,3-O- and 6-O-sulfate groups of heparin. The flexibility of this assay, in both the amount of label incorporated and the variability of solid substrate, makes this an excellent tool to study other heparin binding proteins.
Foxall
C
C
Glycomed Inc., Alameda, California 94501, USA.
Holme
K R
KR
Liang
W
W
Wei
Z
Z
D001667:Binding, Competitive; D001710:Biotin / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry*; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship
D001710:Biotin; D016222:Fibroblast Growth Factor 2; D006493:Heparin; D006497:Heparan Sulfate