An ELISA for selectins based on binding to a physiological ligand

Abstract

Members of the selectin family of adhesion receptors, consisting of L-, P- and E-selectin, mediate the initial interaction between leukocytes and endothelium during leukocyte trafficking from the blood into tissue sites. These receptors have attracted great attention in recent years due to their participation in a number of acute and chronic inflammatory diseases. We describe here a new ELISA that measures the binding between selectin–IgG chimeras and a physiological ligand for L-selectin and can be used to screen selectin inhibitors. The ligand used is a mucin-like glycoprotein known as GlyCAM-1, which is derived from high endothelial venules in secondary lymphoid organs. We demonstrate binding of all three selectins to GlyCAM-1 and demonstrate that the binding interactions satisfy a number of important criteria. The advantage of this ELISA over previous assays is that a macromolecular physiological ligand is employed, rather than a fortuitous or simplified carbohydrate ligand. Thus, the protein–carbohydrate interactions, as well as other interactions contributing to ligand recognition, can be investigated. The assay is suitable for high-throughput screening of compounds and may find use in the identification of selectin antagonists with anti-inflammatory potential.