Clinical Chemistry, Vol 20, 1353-1361, Copyright © 1974 by the American Association for Clinical Chemistry

Radioimmunoassay of Serum Triiodothyronine on Small, Reusable Sephadex Columns

¹ Division of Clinical Pathology, Department of Pathology, School of Medicine, University of California, San Diego, Calif. 92103.

Nicholas M. Alexander, University Hospital, P.O. Box 3548, 225 W. Dickinson St., San Diego, Calif. 92103.

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 (0-100 ng of T₃ per dl). Triiodothyronine concentrations (ng/dl) in human serum are (mean ± SD): euthyroid 96 ± 26, euthyroid females receiving oral contraceptives 132 ± 17, hypothyroid 24 ± 22, and hyperthyroid 263 ± 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 triiodothyronine—albumin (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 ¹²⁵l-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).

Submitted on July 22, 1974
Accepted on August 5, 1974