In vitro receptor binding assays: general methods and considerations

The development of receptor-targeting radiopharmaceuticals commonly involves the use of peptides, antibodies or small molecules. Resulting from the numerous modifications that can be made to these basic targeting agents, research in this field often generates a series of compounds as potential ligands for in vivo investigation. However, measuring each variant of a series in vivo can be both costly and time-consuming. Therefore, a number of in vitro assays, to study interactions between the targeted receptor and the ligands of interest, are frequently used to quickly and inexpensively narrow the field and identify a lead compound(s) for further investigation. For example, in saturation binding studies, the amount of radioligand required to saturate the receptors is measured and analyzed to determine the radioligand equilibrium dissociation constant (Kd), a useful gauge of the receptor binding affinity of a radioligand. In competitive binding experiments, a ligand of interest competes for available receptor sites with a standard radioligand of known high receptor affinity. Competition data are analyzed to yield another indicator of receptor affinity, called an IC50 value, which can be used to rank the relative receptor binding affinities for a series of ligands. In internalization and efflux studies, the rate and extent of receptor-mediated radioligand taken into and subsequently released from cells is measured, providing insight into cellular uptake and retention of the radioligand. Individually or taken together, these in vitro receptor binding assays are useful tools in radiopharmaceutical development.