Ligand dissociation constants from competition binding assays: errors associated with ligand depletion.

The dissociation constant of a ligand at a binding site (e.g., receptor, antibody) is often determined indirectly by competitive displacement of a radioligand. It is well known that such a determination may be seriously in error unless the free concentrations of both the radioligand and the unlabeled ligand can be measured. By means of computer simulations we have explored the conditions under which this error may occur and its magnitude. We offer guidelines for recognizing a probably inaccurate dissociation constant, and we show how, in some cases, a correction can be made. The problem addressed here is not only a theoretical one; it can arise in the ordinary performance of competition binding assays.