Abstract
Using scintillation proximity assay (SPA) technology in combination with Amersham's Leadseeker™ multimodality imaging system we have developed a real-time dissociation kinetics assay to determine the off rates of a panel of antagonists against the chemokine receptor, CCR5. We accomplished this by binding CCR5 membrane preparations to WGA coated polystyrene beads over a period of two hours, incubating with unlabeled antagonists for 5 hours at a concentration 3*Ki, and then initiating the dissociation with a 3H-labeled probe in a 384-well format at 25*Kd. We found that the relevant kinetics could be masked by the use of too much membrane, possibly due to saturation of the bead binding surface; a concentration ratio of 15ug membrane/mg bead was found to be optimum requiring expression levels of receptor of >5.7 pmol/mg. This 384-well assay allows for higher throughput, continuous quantitation as well as the use of less protein, antagonist and radioligand. From these measurements, the experimental data is calculated as the percentage of the receptors bound with the 3H-labeled probe. Dissociation kinetic parameters are estimated by an in-house, web-based statistical tool developed with SAS system software that fits a competitive dissociation model to this data using nonlinear regression.
Published Version
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