The determination of thyroxin (T4) is a basic confirmative test for congenital hypothyroidism. 70% cases of this serioud 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 (107 - 108 М-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 (2mM) 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 TM. Obtained results were within control confidence interval.