Labelled antibody-based one-step time-resolved fluoroimmunoassay for measurement of free thyroxine in serum

Abstract

Background: Valid assays measuring free thyroxine (FT4) must perform without bias despite large variations in the concentrations and affinities of serum thyroxine-binding proteins in the population. We developed a new, rapid one-step labelled-antibody time-resolved fluoroimmunoassay (TRFIA) for FT4. Methods: Based on the heterologous combination of anti-T4 monoclonal antibody and triiodothyronine-immunoglobulin G conjugate, a one-step TRFIA for FT4 detection was established and compared with the two-step DELFIAw Free Thyroxine Assay. Matrix interference caused by endogenous binders and exogenous non-esterified fatty acids (NEFA) was also accessed in the proposed assay. Results: The developed method generally took only one hour, had a detection limit of 0.6 pmol/L and a large linear range of 2.5-120 pmol/L. The inter- and intra-assay coefficients of variation were 3.5-6.6% and 4.4-9.8%, respectively. Results from 110 specimens showed apparent agreement with that from the DELFIAw FT4 Assay with the square of the correlation coefficient of 0.975. This assay indicated that there was no significant dependence on endogenous binders and displayed potential interference by exogenous NEFA up to 5 mmol/L. Conclusions: The proposed one-step heterologous TRFIA FT4 assay possesses simplicity, accuracy, high sensitivity and exhibits great potential for FT4 measurement. The combination of heterologous immunoassay with TRFIA may be advantageous for FT4 immunoassay development.