AN E-SELECTIN BINDING ASSAY BASED ON A POLYACRYLAMIDE-TYPE GLYCOCONJUGATE

Here we show that biotinylated polyacrylamide-type glycoconjugates whi ch contain sialyl Lewis X (sLe(x)-polymer) or sialyl Lewis A (sLe(a)-p olymer) are Ligands for E-selectin, sLe(a)-polymer bound E-selectin wi th higher affinity than sLe(x)-polymer, Based on this property we used the sLe(a)-polymer to establish a sensitive cell-fi ee binding assay for the characterization of E-selectin antagonists, The assay involves complexation of the biotinylated sLe(a)-polymer with streptavidin-per oxidase. This complex is incubated with E-selectin mouse C kappa fusio n protein immobilized onto microtiter plates, found complex is detecte d by the peroxidase reaction. sLe(a)-polymer bound in a Ca2+-dependent manner consistent with the function of E-selectin as a C-typo lectin, Control glycoconjugates with sialic acid (alpha-Neu5Ac), Lewis A (Le( a)), or beta-D-glucose residues instead of sLe(a) failed to interact w ith the E-selectin Neutralizing anti-E-selectin antibodies blocked com pletely binding to E-selectin. This demonstrates specificity of the as say system, sLe(x) blocked binding of the sLe(a)-polymer to E-selectin by 50% at a concentration of 550 mu M (IC50). The assay was used to c haracterize sLe(a)-polymers with differing sLe(a) content as multivale nt inhibitors of E-selectin binding. The inhibitory activity of these polymeric forms of sLe(a) increased with their sLe(a) content up to IC (50)s in the low micromolar range. The binding assay described is sens itive, rapid, and simple and of low variability. Therefore it should b e advantageous fear the identification and characterization of novel E -selectin antagonists, (C) 1996 Academic Press, Inc.