Sensitive time-resolved fluoroimmunoassay for simultaneous detection of serum thyroid-stimulating hormone and total thyroxin with Eu and Sm as labels

Abstract

Based on a novel cocoating strategy and dissociation enhancement lanthanide fluorescence immunoassay technique, a sensitive time-resolved fluoroimmunoassay (TRFIA) has been developed for simultaneous quantification of human serum thyroid-stimulating hormone (TSH) and thyroxin (T4) in a one-and-the-same assay procedure. The new cocoating strategy for preparing highly active surface anti-TSH and anti-T4 monoclonal antibodies (McAbs) was performed by a three-step protocol. Namely, anti-TSH McAb at high concentration (10 μg/ml) and extensively biotinylated bovine serum albumin (BSA) at low concentration (0.5 μg/ml) were coated on microwells by passive adsorption, then streptavidin was captured by the surface BSA–biotin, and finally biotinylated anti-T4 McAb was immobilized by the remnant binding sites of the bound streptavidin. In the present TSH/T4 TRFIA, both sandwich- and competitive-type configurations were involved, and Eu 3+ and Sm 3+ were used as labels for TSH and T4 detection, respectively. The method showed rapid kinetics; the equilibrium was reached within 30 min at 37 °C due to the use of high concentrations of reaction reagents, rapid agitation, and small reaction volume. The lower limits of detection of the method were 0.028 mIU/L for TSH and 4.1 nmol/L for T4 with 20 μL of sample volume. The assay ranges for TSH and T4 were 0.21–80.00 mIU/L and 20–300 nmol/L, respectively. The correlation between the TSH/T4 values obtained by the present TSH/T4 TRFIA and those obtained by commercial chemiluminescence immunoassay was satisfactory.