Here we show that biotinylated polyacrylamide-type glycoconjugates which contain sialyl Lewis X (sLex-polymer) or sialyl Lewis A (sLea-polymer) are ligands for E-selectin. sLea-polymer bound E-selectin with higher affinity than sLex-polymer. Based on this property we used the sLea-polymer to establish a sensitive cell-free binding assay for the characterization of E-selectin antagonists. The assay involves complexation of the biotinylated sLea-polymer with streptavidin-peroxidase. This complex is incubated with E-selectin mouse Ckappa fusion protein immobilized onto microtiter plates. Bound complex is detected by the peroxidase reaction. sLea-polymer bound in a Ca2+-dependent manner consistent with the function of E-selectin as a C-type lectin. Control glycoconjugates with sialic acid (alpha-Neu5Ac), Lewis A (Lea), or beta-D-glucose residues instead of sLea failed to interact with the E-selectin. Neutralizing anti-E-selectin antibodies blocked completely binding to E-selectin. This demonstrates specificity of the assay system. sLex blocked binding of the sLea-polymer to E-selectin by 50% at a concentration of 550 microM (IC50). The assay was used to characterize sLea-polymers with differing sLea content as multivalent inhibitors of E-selectin binding. The inhibitory activity of these polymeric forms of sLea increased with their sLea content up to IC50s in the low micromolar range. The binding assay described is sensitive, rapid, and simple and of low variability. Therefore it should be advantageous for the identification and characterization of novel E-selectin antagonists.