Time-resolved fluorescence immunoassay of thyroxine in serum: immobilized antigen approach

With T(4)-bovine IgG as a solid-phase antigen, we have developed a direct competitive-type immunoassay for serum total thyroxine (TT(4)), which depends on the competitive distribution of europium-labeled anti-T(4) monoclonal antibody between solid-phase-bound T(4) and the T(4) in the sample or standard. The captured fraction of the tracer was measured after a dissociation-enhancement step. Four different T(4) protein conjugates were synthesized, of which T(4)-bovine IgG was selected as the most favorable for the preparation of solid-phase antigen. The sensitivity was 3.5 ng/ml with a sample volume of 20 microl. T(4) values obtained by this procedure agreed well with those obtained by RIA (r = 0.967, n = 38) and EG&G Wallac TRFIA (r = 0.926, n = 64). All other quality criteria was also fulfilled with respect to precision, accuracy, and dynamic range.