A sensitive radioimmunoassay for measurements of 3,3'-diiodothyronine in unextracted serum

Abstract

A radioimmunoassay for measurements of 3,3'-diiodothyronine (3,3'-T2) in unextracted serum is described. Antisera to 3,3'-T2 were produced by immunization of rabbits with 3,3'-T2 coupled to human serum albumin. Scatchard plot analysis of the chosen antiserum gave an effective equilibrium constant of 1.1 × 10¹¹l/mol. The 3,3'-T2 binding capacity in undiluted serum was calculated to be 9.1 μmol/l. Monolabelled (¹²⁵I)3,3'-T2 having a specific activity of 3800 mCi/mg was synthetized from 3-monoiodothyronine. The lowest concentration of standard inducing a consistent fall in per cent bound (¹²⁵I)3,3'-T2 was 6.0 pmol/l (0.24 fmol/tube). Triiodothyronine (T3) cross-reacted significantly in the assay. At 50% inhibition of binding of (¹²⁵I)3,3'-T2, the cross-reaction was 0.29% (mol/mol), but at the low physiological concentrations of T3 it amounted to approximately 1%. T3 cross-reaction was corrected for in every assay. The interference of 3,3'-T2 binding proteins in serum was precluded by adding 8-anilino-l-napthalenesulphonicacid(ANS). Mean 3,3'-T2 concentrations were 38.9 pmol/l in fourteen normal young females and 48.9 pmol/l in fifteen normal young males. When 3,3'-T2 was measured (1) in ethanol extracts of serum, (2) in assays where T3 was added to standards in amounts equivalent to those present in the samples and T3 correction omitted, and (3) using a different 3,3'-T2 antibody, similar levels of 3,3'-T2 were found in normal serum.

Key Words:

• anilino-napthalene sulphonic acid (ANS)
• wick chromatography