DEVELOPMENT OF SCINTILLATION PROXIMITY ASSAYS FOR E-SELECTIN AND THEIR APPLICATION IN TESTING POTENTIAL ANTAGONISTS

E-selectin, a cell adhesion molecule expressed on endothelial cells, i s 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/act ivity relationship of these weak affinity antagonists, we have develop ed several scintillation proximity assays (SPAs) for E-selectin. Two o f these involve immobilizing E-selectin to streptavidin-coated SPA bea ds through a biotinylated anti-E-selectin monoclonal antibody. These b eads are incubated with I-125-labeled carcinoembryonic antigen (an sle (x)-containing protein) or H-3-labeled HL-60 cells and the amount of b ound ligand is quantitated by counting in a beta-scintillation counter . In addition, we have developed a method to prepare a functionally ac tive biotinylated E-selectin, which can be directly coupled to SPA bea ds and assayed for ligand binding. These SPAs are sensitive, reproduci ble, and suitable for screening antagonists and studying structure/act ivity relationships of lead compounds. By using the SPA, we have also showed that an sLe(x) polyacrylamide polymer is 10 times more potent t han the monomer, suggesting that the potency of E-selectin antagonists can be greatly enhanced by multivalent presentation. (C) 1995 Academi c Press, Inc.