Analysis for Total Serum Thyroxine by Equilibrium Competitive Protein Binding on Small, Re-usable Sephadex Columns

Abstract

Abstract We describe a modified method of analysis for total thyroxine in 50 µl of serum by equilibrium competitive protein binding with thyroxine-binding globulin. Columns (9 x 23 mm) are used containing Sephadex G-25, which are regenerated after each cycle by removing residual thyroxine adsorbed to the gel with excess thyroxine-binding globulin (dilute plasma). As little as 1 ng of hormone can be detected, and recoveries of added thyroxine from sera range from 98-102%. For either between-assay or within-assay determinations, the coefficients of variation are about 4%. Results of this method as compared with those for the uncorrected Murphy-Pattee procedure [J. Lab. Clin. Med. 66, 161 (1965)] gave a correlation coefficient of 0.944, but results with the present procedure are 11% higher because of incomplete and variable recovery with the uncorrected Murphy-Pattee method. Our procedure offers the advantage that, on a single column, thyroxine is quantitatively extracted from serum, rapidly brought to equilibrium binding with thyroxine binding globulin, and the free and bound thyroxine are separated. Cumbersome and lengthy procedures of extraction, centrifugation, and evaporation are thus obviated.