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[Lanthanide fluorescent immune analysis for determination of hormone thyroxine in dry blood spots]
V V Zherdeva1, A V Chudinov, A P Savitskiĭ
1Bakh Institute of Biochemistry Russian Academy of Sciences, 119071 Moscow, Leninsky pr., 33. vjerdeva@inbi.ras.ru
Insights
This study developed a new immunofluorescent assay for thyroxin (T4) in dried blood spots, improving congenital hypothyroidism screening. The assay offers accurate T4 detection, aiding early diagnosis of this often asymptomatic disease.
Area of Science:
- Biochemistry
- Immunology
- Neonatal Screening
Context:
- Congenital hypothyroidism (CH) diagnosis relies on thyroxin (T4) testing.
- CH often presents asymptomatically, necessitating sensitive detection methods.
- Dried blood spot (DBS) analysis is crucial for neonatal screening programs.
Purpose:
- To develop and validate a novel immunofluorescent T4 assay for DBS.
- To utilize anti-T4 monoclonal antibodies and europium chelate for enhanced detection.
- To establish a reliable and accurate method for T4 determination in CH screening.
Summary:
- A new immunofluorescent T4 assay was developed using anti-T4 monoclonal antibodies and europium chelate (DA-DTPA).
- The assay demonstrated a T4 detection limit of 10 nM with a variation coefficient under 15%.
- Monoclonal antibody characterization (affinity, cross-reactivity) and assay optimization (buffer additives) were performed, with results validated against a control kit.
Impact:
- Provides a sensitive and accurate method for T4 measurement in DBS, crucial for CH diagnosis.
- Enhances the capability of neonatal screening programs to detect CH effectively.
- Contributes to the early identification and management of CH, preventing long-term health issues.
Abstract:
The determination of thyroxin (T4) is a basic confirmative test for congenital hypothyroidism. 70% cases of this period inborn disease are taking asymptomatic course. We developed immunofluorescent T4-assay in dried blood spots with anti-T4 monoclonal antibody and europium chelate (dianhydride of diethylentriaminipentaacetic acid (DA-DTPA)). This method requires DELFIA Plate Fluorometer 1232 (Wallac, Finland) or its sub modifications. Panel of monoclonal antibodies for T4 has been obtained. Type/subtype (IgG2b, IgG1), affinity constants (10(7)-10(8) M-1), cross reactivity to homologous structures (0-2%) were determined. Stable clones with high affinity and viability were selected for the development of the assay. Conjugates of thyroxin and europium chelate were synthesized. Inclusion of sodium salicylate (2 mg/ml) and EDTA (2 mM) into the buffer reduced the nonspecific signal. Limit for T4 detection (T4 standarts) was 10 nM with not more than 15% variation coefficient. Accuracy was estimated by Bio-Rad Lipochek Immunoassay Plus Control Kit. Obtained results were within control confidence interval.
