Radioimmunoassay for serum triiodothyronine: evaluation of simple techniques to control interference from binding proteins.

Simple techniques for controlling interference from binding proteins in serum, such as thyroxine-binding globulin, in radioimmunoassay for triiodothyronine (13) 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 13 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 g/100 d of serum) might be too low to displace 13 from all its binding sites, but a concentration of ANS greater than 200 ig/100 tI of serum interferes with 13 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 13 binding.