PMID: 8600819Nov 20, 1995Paper

Development of scintillation proximity assays for E-selectin and their application in testing potential antagonists

Analytical Biochemistry
M Anostario, K S Huang

Abstract

E-selectin, a cell adhesion molecule expressed on endothelial cells, is involved in the trafficking of neutrophils to areas of inflammation. The tetrasaccharide sialyl Lewis x (sLe(x)) and other analogues have been shown to be weak affinity antagonists. To study the structure/activity relationship of these weak affinity antagonists, we have developed several scintillation proximity assays (SPAs) for E-selectin. Two of these involve immobilizing E-selectin to streptavidin-coated SPA beads through a biotinylated anti-E-selectin monoclonal antibody. These beads are incubated with 125I-labeled carcinoembryonic antigen (an sLe(x)-containing protein) or 3H-labeled HL-60 cells and the amount of bound ligand is quantitated by counting in a beta-scintillation counter. In addition, we have developed a method to prepare a functionally active biotinylated E-selectin, which can be directly coupled to SPA beads and assayed for ligand binding. These SPAs are sensitive, reproducible, and suitable for screening antagonists and studying structure/activity relationships of lead compounds. By using the SPA, we have also showed that an sLe(x) polyacrylamide polymer is 10 times more potent than the monomer, suggesting that the potency of E-selectin ...Continue Reading

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