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-bindi ng growth factor family that interacts with cell surface heparan sulfa te (HS) proteoglycans and extracellular matrix heparin. Here we report the development of a simple and sensitive assay that used biotinylate d HS or heparin to bind to bFGF coated onto 96-well microtiter plates. Bound labeled HS or heparin was reacted with enzyme-linked streptavid in and results were recorded as optical density. Increased molar exces s of biotin resulted in increased incorporation of biotin and higher s ignal without compromising binding. Glycosaminoglycans and modified he parins 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 sulfat e demonstrated the specificity of the glycosaminoglycan binding. Struc tural 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 moderat e changes in binding capacity. Results from this assay are in agreemen t with previous data from our laboratory and reports from other resear chers 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 fle xibility of this assay, in both the amount of label incorporated and t he variability of solid substrate, makes this an excellent tool to stu dy other heparin binding proteins. (C) 1995 Academic Press, Inc.