Miniaturized receptor binding assays: complications arising from ligand depletion.
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Carter, Clare, Leighton-Davies, Juliet and Charlton, Steven (2007) Miniaturized receptor binding assays: complications arising from ligand depletion. Journal of Biomolecular Screening : the official journal of the Society for Biomolecular Screening, 12 (2). pp. 255-266. ISSN 1087-0571
Abstract
The advent of miniaturized assay formats has made possible the screening of large numbers of compounds against a single target, known as high-throughput screening. Despite this clear advantage, assay miniaturization also increases the risk of ligand depletion, where the actual concentration of free ligand is significantly lower than that added. This, in turn, complicates the interpretation of data from such assays, potentially introducing significant error if not recognized. In this study, the effects of reducing assay volume on radioligand Kd and competitor Ki values have been investigated, using the muscarinic M(3) receptor as a model system. It was found that assay miniaturization caused dramatic effects, with up to a 30-fold underestimation of ligand affinity. A theoretical model was developed and shown to accurately predict both the degree of ligand depletion in any given assay volume and the effect of this depletion on affinity estimates for competing ligands. Importantly, it was found that in most cases, errors introduced by ligand depletion could be largely corrected for by the use of appropriate analysis methods. In addition to those previously described by others, the authors propose a simple method capable of correcting errors in competition binding experiments performed in conditions of ligand depletion.
| Item Type: | Article |
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| Additional Information: | author can archive post-print (ie final draft post-refereeing); Publisher's version/PDF cannot be used |
| Keywords: | ligand depletion; binding assay; miniaturization; radioligand |
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| Date Deposited: | 14 Dec 2009 13:58 |
| Last Modified: | 14 Dec 2009 13:58 |
| URI: | https://oak.novartis.com/id/eprint/539 |