Construction and use of a RIA-gram

All equilibrium protein binding assays, as exemplified by radioimmunoassays (RIA) are governed by the law of mass action. If the reactants are homogenous, then the reaction is characterized by a simple apparent dissociation constant (Kd). We have calculated a set of binding curves, a RIA-gram, in which all concentrations are expressed with Kd as unit. This RIA-gram can be generally employed to optimise equilibrium protein binding assays. Using the RIA-gram we illustrate three points. First, the sensitivity of a given assay is related to either the Kd or the concentration of the labeled ligand, whichever is the larger. Second, if Kd determines the sensitivity of the assay, one can not improve the sensitivity by decreasing the concentration of the labeled ligand below 0.1Kd. Finally, in order to obtain an optimal assay the fraction of ligand bound when only labeled ligand is present depends on the concentration of labeled ligand and on the concentration for which one wants the highest sensitivity of the assay.