FIGURE 6.

IC₅₀ values for the binding of HS antibodies to PMHS in the presence of competing chemically modified heparins, unmodified heparin and HS. PMHS was biotinylated and immobilized on streptavidin-coated microtiter plates. Equilibrium binding of HS antibodies in the presence of variable concentrations of heparin, HS, and modified heparin derivatives was quantified at A₄₉₀ using a VSV-G tag antibody (P5D4) followed by HRP-conjugated anti-mouse IgG and o-phenylenediamine substrate. Modified heparins are: de-N-sulfated/N-acetylated heparin (NAc), de-2-O-sulfated heparin (de2O), de-6-O-sulfated heparin (de6O), de-N-sulfated/N-acetylated and de-2-O-sulfated heparin (de2ONAc), de-N-sulfated/N-acetylated and de-6-O-sulfated heparin (de6ONAc), exclusively N-sulfated heparin (NS only), and completely de-O/N-sulfated/N-acetylated (de2,6ONAc) IC₅₀ values were then calculated as the concentration of competitor saccharide needed for 50% inhibition of antibody binding to immobilized HS. The values are the means of triplicate samples, the error bars represent the S.E., and the data are representative of two separate experiments. * denotes oligosaccharides unable to compete sufficiently to calculate an IC₅₀.