Optimization of T assay: a model study

Factors involved in the optimization of competitive binding radioassays have been analyzed using a thyroxine (T4) radioassay as a model system. The effects of kinetic errors are minimized by the use of the lowest possible incubation temperature for the separation step and by the use of labeled T4 of high purity (which is not consistently available from commerical sources). The use of labeled T4 containing significant quantities of labeled triiodothyronine (T3) can lead to a marked increase in bias and a decrease in precision with relatively small errors in the separation step due to the relatively short half-life of the T3-binder complex. The determination of the dissociation rate constant (not to be confused with the affinity constant) allows one to make useful estimates of the tolerances that can be allowed in the separation step in order to achieve a desired level of accuracy and precison.