Development of two-step RIA for the measurement of free triiodothyronine in human serum

Development of a simple, user friendly and valid two step radioimmunoassay format based on antibody coated tubes for the measurement of free triiodothyronine (FT3) in human serum. The system uses a polyclonal anti-T3 antibody with an affinity constant of 0.6 × 10¹¹ L/mol. Radiolabelled triiodothyronine was prepared by radiolabelling diiodothyronine with ¹²⁵I by chloramine-T oxidation method and purified over Sephadex G-25 column. FT3 standards of concentrations ranging from 0 to 30 pg/mL were prepared in T3 free human serum. 0.14 M Tris-saline buffer pH 7.4 and 0.035 M Tris-saline buffer pH 7.4 with 0.1% Tween-20 were used as assay buffer and wash buffer respectively. Assay covers a range of 0-30 pg/mL with the sensitivity of 0.4 pg/mL. Anti-T3 antibody concentration, reaction time, reaction volume and sample volume were optimised to yield 1% extraction efficiency which is mandatory for a valid free hormone measurement. Analytical assay parameters such as intra assay variation and inter assay variation were within acceptable limits. Recoveries at FT3 concentrations 1.5, 2.8 and 4.8 pg/mL were 90, 102 and 92.5% respectively. Progressive dilution of hypo and euthyroid samples up to 1:96 times yielded virtually constant values validating the assay procedure, whereas a hyperthyroid sample with FT3 concentration of 9.9 pg/mL declined to 60% at 1:96 times dilution as expected. Euthyroid range established using the developed system is 1.6-4pg/mL. The FT3 concentration of clinical samples measured using the optimised procedure correlated well with the commercial kit value (r = 0.94). Simple, user friendly and valid immunoassay procedure developed as per the guidelines of American Thyroid Association can be used for the routine measurement of free triiodothyronine in human serum. (author)