Abstract
The focus of this review paper is factors affecting data interpretation in ligand binding assays under equilibrium conditions. Protocols for determining K(d) (the equilibrium dissociation constant) and K(dA) (the equilibrium inhibitor constant) for receptor ligands are discussed. The basic theory describing the interaction of a radiotracer and an unlabelled competitor ligand with a receptor is developed. Inappropriate experimental design may result in ligand depletion and non-attainment of equilibrium, distorting the calculation of K(d) and K(dA) . Strategies, both theoretical and practical, will be given to avoid and correct such errors, thus leading to the determination of reliable values for these constants. In determining K(dA) from competition binding studies, two additional concepts are discussed. First, the necessity to measure an adequate specific binding signal from the bound radiotracer ligand limits the range of affinity constants that can be measured: a particular set of assay conditions may lead to an upper limit on the apparent affinity of unlabelled ligands. Second, an extension of the basic assay methodology can indicate whether the interaction between the tracer and a test ligand is mediated by a competitive or an allosteric mechanism. Finally, the review ends with a discussion of two factors that are often overlooked: buffer composition and the temperature at which the assay is conducted, and the impact these can have on affinity measurements and the understanding of drug interactions.