Exp Clin Endocrinol Diabetes 2013; 121 - P13
DOI: 10.1055/s-0033-1336705

Establishment of a competitive chemiluminescence immunoassay to detect 3,5-T2 in human serum

I Lehmphul 1, Z Wu 2, CJ Strasburger 2, J Koehrle 1
  • 1Charité – Universitätsmedizin Berlin, Institut für Experimentelle Endokrinologie, Berlin, Germany
  • 2Charité – Universitätsmedizin Berlin, Department of Medicine for Endocrinology, Diabetes and Nutritional Medicine, Berlin, Germany

Introduction: 3,5-diiodothyronine (3,5-T2), a potential metabolite of the thyroid hormone (TH) 3,3',5-triiodothyronine (T3), exerts important catabolic action without undesirable side effects. 3,5-T2 supports loss of body fat by increasing energy expenditure while lean muscle constitution is maintained. An immunoassay based quantification of 3,5-T2 in human serum may provide a fast and reproducible method for further insight into the relation between 3,5-T2 serum levels and body composition.

Methods: Anti-3,5-T2 monoclonal antibodies (3,5-T2 mAbs) were produced with the hybridoma technology and selected according to their high affinity to 3,5-T2 and no reactivity to BSA, to which the hapten was conjugated. The specificity of selected 3,5-T2 mAbs was tested by determining its cross reactivity to structurally related TH and thyronamines (TAM). We selected one monoclonal antibody, developed a competitive immunoassay and optimized it for high sensitivity, linearity and recovery.

Results: Four 3,5-T2 mAbs (9D3, 9G11, 2D2 and 4F8) showed nearly no cross reactivity to the tested TH 3,3'-T2, 3',5'-T2, T3, T4, rT3 (below 0.1%). Furthermore, mAbs 2D2 and 4F8 had lowest cross reactivity (1 – 2%) to 3,5-T2AM and diiodothyroacetic acid. MAb 4F8 was used to construct the competitive immunoassay. The assay detects 3,5-T2 levels in human serum within a range of 0.3 – 5nM with a lower detection limit of 0.24nM 3,5-T2 (n = 24). A recovery of 98.2 ± 6.1% and 97.2 ± 3.5% was obtained after addition of 0.2nM or 1nM 3,5-T2 to serum samples. Linearity upon 2- or 4-fold dilution of the same human serum samples showed a recovery of 107.3 ± 1.8% and 126.2 ± 15.4% respectively (mean ± SD%).

Conclusion: So far the immunoassay showed high specificity, sensitivity and accuracy for 3,5-T2 detection. Parameters obtained so far are promising that this new mAb based competitive immunoassay will provide a useful analytical method to quantify 3,5-T2 in human serum.

Supported by DFG GK1208 – 2 TP3 & TP8.