Radioimmunoassay for Serum Triiodothyronine: Evaluation of Simple Techniques to Control Interference from Binding Proteins

Abstract

Simple techniques for controlling interference from binding proteins in serum, such as thyroxine-binding globulin, in radioimmunoassay for triiodothyronine (T₃) have been evaluated for their efficacy, and their effect on assay sensitivity and on recovery of added T₃. Ethanol precipitation of serum proteins decreased the assay sensitivity, nonspecific binding was increased, and recoveries of added T₃ were inconsistent. Heat-inactivation of thyroxine-binding globulin or use of 8-anilino-1naphthalene sulfonic acid (ANS) to displace T₃ from thyroxine-binding globulin produced comparable recovery rates. The heat-inactivation method slightly decreased the sensitivity of the assay and prolonged the procedure, whereas use of ANS is simple, and the assay sensitivity is maintained. When sera contain a high concentration of thyroxine-binding globulin, a fixed concentration of ANS (175 µg/100 µl of serum) might be too low to displace T₃ from all its binding sites, but a concentration of ANS greater than 200 µg/100 µl of serum interferes with T₃ quantitation by competitively binding to the antibodies. The cross-reactivity of thyroxine to T₃-antibodies varies with the antiserum. Thyroxine-binding globulin appears to be the only protein in serum that competes with the antibody for T₃ binding.