title: Selectin/glycoconjugate binding assays for the identification and optimization of selectin antagonists.
abstract: In this study we describe ELISA-type P- and L-selectin binding assays for the analysis of selectin antagonists. A biotinylated polyacrylamide-type glycoconjugate containing sialyl Lewis A (sLe(a)-polymer) is utilized as a synthetic ligand for both selectins analogous to the E-selectin assay we have developed recently. Following precomplexation of sLe(a)-polymer with streptavidin-peroxidase, the complex is added to microtiter plates coated with the recombinant selectins. Binding of sLe(a)-polymer to the immobilized selectins is measured by the peroxidase reaction. SLe(a)-polymer was found to bind to P- and L-selectin in a cation-dependent manner. The interaction of the polymer was blocked by neutralizing anti-P- and anti-L-selectin antibody, respectively. The reference compounds heparin and fucoidan inhibited in both assays. Sialyl Lewis X (sLe(x)) blocked binding to L-selectin by 46% at 3 mM, whereas no inhibition was observed in the P-selectin assay up to 3 mM. Control polymers containing sialic acid or beta-d-glucose instead of sLe(a) weakly bound or failed to bind to the selectins. Both assays are rapid to perform and of low variability. The P-selectin assay was successfully employed to identify and optimize novel carbohydrate-based P-selectin antagonists. The P-, L-, and E-selectin assays were used to determine the fine selectivity of several sLe(x)-related selectin antagonists. These studies together suggest that sLe(a)-polymer-based selectin assays are well suited for primary screening and the characterization of selectin antagonists.
authors.affiliation: Transplantation Research, Novartis Pharma A.G., Basel, CH-4002, Switzerland.
authors.forename: G
authors.initials: G
authors.lastname: Weitz-Schmidt
authors.forename: K W
authors.initials: KW
authors.lastname: Gong
authors.forename: C H
authors.initials: CH
authors.lastname: Wong
mesh_terms: D000818:Animals; D018395:CA-19-9 Antigen; D016466:CHO Cells; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D018922:HL-60 Cells; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D019041:L-Selectin / Q000378:metabolism*; D008956:Models, Chemical; D009844:Oligosaccharides / Q000378:metabolism*; D019007:P-Selectin / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D011134:Polysaccharides / Q000494:pharmacology; D011485:Protein Binding; D000080506:Sialyl Lewis X Antigen
chemical_list: D018395:CA-19-9 Antigen; D009844:Oligosaccharides; D019007:P-Selectin; D011108:Polymers; D011134:Polysaccharides; D000080506:Sialyl Lewis X Antigen; D019041:L-Selectin; D006493:Heparin; C007789:fucoidan