Radioimmunoassay of serum triiodothyronine on small, reusable, Sephadex columns.

We have developed a radioimmunoassay method for serum triiodothyronine on small (9 X 23 mm), reusable Sephadex G-25 columns. Recoveries of triiodothyronine added to sera are 97-105%, and the coefficients of variation for within-assay and between-assay determinations are 5.8 and 9.0 % , respectively. Maximum sensitivity is in the hypothyroid range (0100 ng of 13 per dl). Triiodothyronine concentrations (ng/dl) in human serum are (mean ± SD): euthyroid 96 ± 26, euthyroid females receiving oral contraceptives 132 ± 17, hypothyroid 24 :1: 22, and hyperthyroid 263 L 58. Mild hemolysis and lipemia do not interfere, but 10-20 % artifactual elevation is found with icteric sera. Interference by thyroxine is negligible. An improved, reproducible carbodiimide coupling procedure for triiodothyroninealbumin (19:1) antigen is described. A parenthetic observation of potential importance is that immunized rabbits accumulate large concentrations (30 000-90 000 ng/dl) of serum bound triiodothyronine. Our procedure offers the advantages that, on a single column, triiodothyronine is quantitatively extracted from serum that is premixed with 1251-labeled triiodothyronine, serum proteins are removed, binding with antiserum is accomplished in 1 h, and the free and bound triiodothyronine are separated. The columns are regenerated for repetitive use by eluting residual iodothyronines with excess thyroxine-binding globulin (dilute plasma).