Selectin/glycoconjugate binding assays for the identification and optimization of selectin antagonists

In this study we describe ELISA-type P- and L-selectin binding assays for t he analysis of selectin antagonists. A biotinylated polyacrylamide-type gly coconjugate containing sialyl Lewis A (sLe(a)-polymer) is utilized as a syn thetic ligand for both selectins analogous to the E-selectin assay we have developed recently. Following precomplexation of sle(a)-polymer with strept avidin-peroxidase, the complex is added to microtiter plates coated with th e recombinant selectins. Binding of sLe(a)-polymer to the immobilized selec tins is measured by the peroxidase reaction. SLe(a)-polymer was found to bi nd to P- and L-selectin in a cation-dependent manner. The interaction of th e polymer was blocked by neutralizing anti-P- and anti-L-selectin antibody, respectively. The reference compounds heparin and fucoidan inhibited in bo th 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 t o perform and of low variability. The P-selectin assay was successfully emp loyed to identify and optimize novel carbohydrate-based P-selectin antagoni sts. The P-, L-, and E-selectin assays were used to determine the fine sele ctivity of several sLe(x)-related selectin antagonists. These studies toget her suggest that sLe(a)-polymer-based selectin assays are well suited for p rimary screening and the characterization of selectin antagonists. (C) 1999 Academic Press.