Assay performance and tracer properties for two analog-based assays of free triiodothyronine.

We determined binding characteristics of the triiodothyronine (T3) analog tracer used in the Amerlex and Amerlex-M FT3 radioimmunoassay for the three endogenous binding proteins in serum: thyroxin-binding globulin (TBG), thyroxin binding prealbumin (PA), and albumin. Both T3 and its analog bind to the same sites on TBG and PA. However, the analog has significantly lower association constants (1.0% and 3.8%, respectively, of T3 binding affinity) and it binds to different sites on albumin. Analog binding is characterized by two (weak) specific binding sites [K = 0.46 (SD 0.03) X 10(5) L/mol]; T3 is bound at about 28 very weak, nonspecific sites [K = 0.41 (SD 0.03) X 10(4) L/mol]. Sera from healthy subjects with a wide range of concentrations of binding proteins showed no interference from analog binding in the FT3 assay. In contrast, in vitro studies of albumin binding revealed a weak dependence of both assays on albumin concentration (0.05 pmol of FT3 per gram of albumin per liter), an interference probably unimportant for most laboratory samples. Nonesterified fatty acids (NEFA) and the T3 analog apparently bind to different sites on albumin; thus the Amerlex FT3 assay is insensitive to moderately increased concentrations of NEFA in serum.