Influence of Adsorption and Deproteination on Potential Free Thyroxine Reference Methods

Abstract

Abstract Background: There is a need for consensus concerning reference methods to be used for calibration of commercial free-thyroxine (FT4) assays. Methods: Three different potential reference techniques were investigated for adsorption of T4 to membrane materials, including any in vitro solid surfaces to which T4 might adsorb, and for efficient separation of the T4-binding proteins from the free hormone fraction. We compared ultrafiltration with different commercial ultrafiltration units, ultrafiltration by dialysis tubing, equilibrium dialysis, and ultracentrifugation. We measured the adsorption to membranes and materials with l-[125I]T4. Separation efficiency was determined by measuring the T4-binding protein albumin in the ultrafiltrate and the dialysate as well as in the supernatant from the ultracentrifugation with a double-antibody sandwich ELISA technique. Results: We found a constant relationship between the amount of T4 adsorbed to the dialysis or ultrafiltration membranes/materials and the initial T4 concentration in HEPES buffer (protein-free medium). T4 was considerably less adsorbed from serum than from HEPES buffer (P <0.001). Serum T4 was less adsorbed upon ultracentrifugation than during dialysis and ultrafiltration (P <0.001). It was difficult to completely separate FT4 from the binding proteins by ultrafiltration and ultracentrifugation. Separation by ultrafiltration depended on the material used. Conclusions: No investigated separation technique provides technically and theoretically correct separation of the free fraction of T4 from the protein-bound fraction. Equilibrium dialysis seems to be the least compromised.