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Real‐time measurement of dissociation rates of unlabeled compounds from a membrane receptor
Author(s) -
Gee Peter David,
Beavis Paul,
Lee Ina,
Ravindran Palanikumar,
Hu Steven,
Swinney David
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a919
Subject(s) - chemistry , kinetics , dissociation (chemistry) , membrane , radioligand , receptor , receptor–ligand kinetics , chromatography , biophysics , biochemistry , organic chemistry , biology , physics , quantum mechanics
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 3 H‐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 3 H‐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.