Homogeneous time-resolved fluoroimmunoassay of 3,5,3′-triiodo-l-thyronine in human serum by using europium fluorescence energy transfer

A sensitive homogeneous time-resolved fluoroimmunoassay method for 3,5,3′-triiodo-l-thyronine (T3) based on the fluorescence resonance energy transfer (FRET) from a fluorescent Eu3+ complex, {[(4,6-dichloro-1,3,5-triazin-2-yl)amino-biphenyl-4‴-yl]-2,2′:6′,2″-terpyridine-6,6″-diyl}bis(methylenenitrilo) tetrakis(acetate)-Eu3+ (DTBTA-Eu3+) (λex,max = 335 nm, λem,max = 615 nm), to an organic fluorescence dye Cy5 has been developed. The new assay system combined the use of DTBTA-Eu3+-labeled T3-bovine serum albumin (BSA) conjugate and Cy5-labeled anti-T3 monoclonal antibody for a competitive-type immunoassay. After the competitive reactions of DTBTA-Eu3+-labeled T3-BSA and T3 sample with Cy5-labeled anti-T3 antibody, the T3 concentration was measured with a time-resolved mode by monitoring the sensitized emission of Cy5 derived from FRET in a homogeneous format. The method gives the detection limit of 0.26 ng/ml. The coefficient variations of the method are less than 2.0% and the recoveries are in the range of 80–111% for serum sample measurement. The concentrations of T3 in 30 human serum samples were determined, and the results were compared with those of the independently determined by a radio-immunoassay method. A good correlation was obtained with a correlation coefficient of 0.989.