Sensitive time-resolved fluoroimmunoassay for simultaneous detection of serum thyroid-stimulating hormone and total thyroxin with Eu and Sm as labels.
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 micro g/ml) and extensively biotinylated bovine serum albumin (BSA) at low concentration (0.5 micro g/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 30min at 37 degrees 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 micro L 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.
Wu
Feng Bo
FB
Department of Isotope, China Institute of Atomic Energy, P.O. Box. 275-39, 102413, Beijing, People's Republic of China. wufb@etang.com
Han
Shi-Quan
SQ
Xu
Tao
T
He
You-Feng
YF
D005063:Europium / Q000032:analysis*; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D007700:Kinetics; D012493:Samarium / Q000032:analysis*; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine; D013194:Staining and Labeling; D013972:Thyrotropin / Q000097:blood* / Q000276:immunology; D013974:Thyroxine / Q000097:blood* / Q000276:immunology; D013997:Time Factors
D012710:Serum Albumin, Bovine; D012493:Samarium; D005063:Europium; D013972:Thyrotropin; D013974:Thyroxine