Calculating specific binding for prolactin receptor assays.

Specific binding of [125I]iodo-prolactin and receptor is usually calculated by subtracting the amount of bound radioactivity of assays containing excess unlabeled hormone (competed) from that of noncompeted assays. A proportional method to calculate specific binding of prolactin-receptor or other hormone-receptor interactions is introduced as a replacement for the currently used subtraction method. The proportional method calculates specific binding by multiplying the ratio of free radioactivities of noncompeted and competed assays by the radioactivity retained in the competed assay and subtracting this value from the radioactivity retained by the noncompeted assay. In prolactin-receptor assays greater specific binding is seen when data is calculated by this proportional method rather than the subtraction method. The amount of this increase relates directly to the levels of specific and nonspecific binding. Additionally, the binding characteristics of prolactin and receptor in both equilibrium and kinetic studies are significantly different when specific binding is calculated by the proportional method rather than the subtraction method.