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Sensitive time-resolved fluoroimmunoassay for simultaneous detection of serum thyroid-stimulating hormone and total
Feng Bo Wu1, Shi-Quan Han, Tao Xu
1Department of Isotope, China Institute of Atomic Energy, P.O. Box. 275-39, 102413, Beijing, People's Republic of China. wufb@etang.com
Insights
A new time-resolved fluoroimmunoassay (TRFIA) enables simultaneous measurement of thyroid-stimulating hormone (TSH) and thyroxin (T4) using a novel cocoating strategy. This method offers sensitive and rapid quantification of these critical thyroid hormones in human serum.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Immunology
Background:
- Accurate quantification of thyroid hormones like TSH and T4 is crucial for diagnosing thyroid disorders.
- Existing immunoassays may lack the sensitivity or efficiency for simultaneous, rapid analysis.
Purpose of the Study:
- To develop a sensitive time-resolved fluoroimmunoassay (TRFIA) for the simultaneous quantification of human serum TSH and T4.
- To implement a novel cocoating strategy to enhance assay performance and efficiency.
Main Methods:
- A three-step cocoating strategy was employed to immobilize anti-TSH and anti-T4 monoclonal antibodies (McAbs) onto microwells.
- Utilized Eu(3+) and Sm(3+) as labels for TSH and T4 detection, respectively, within a combined sandwich- and competitive-type assay configuration.
- Optimized reaction conditions including high reagent concentrations, rapid agitation, and small reaction volumes for rapid kinetics.
Main Results:
- Achieved lower limits of detection of 0.028 mIU/L for TSH and 4.1 nmol/L for T4.
- Established assay ranges of 0.21-80.00 mIU/L for TSH and 20-300 nmol/L for T4.
- Demonstrated satisfactory correlation with commercial chemiluminescence immunoassay methods.
Conclusions:
- The developed TSH/T4 TRFIA is a sensitive and rapid method for simultaneous thyroid hormone quantification.
- The novel cocoating strategy effectively enhances antibody immobilization and assay performance.
- This assay provides a reliable alternative for clinical diagnostics requiring simultaneous TSH and T4 measurement.
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 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.
