Tanja Diana1, Yunsheng Li2, Paul D Olivo2, Karl J Lackner3, Hannah Kim2, Michael Kanitz1, George J Kahaly1. 1. 1 Molecular Thyroid Research Laboratory, Johannes Gutenberg University Medical Center , Mainz, Germany . 2. 2 Research & Development, Quidel Corporation, San Diego, California. 3. 3 Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Medical Center , Mainz, Germany .
Abstract
BACKGROUND AND OBJECTIVE: A cell-based bioassay for the measurement of thyroid blocking autoantibodies (TBAb) has been recently reported. The analytical performance and validation of this bioassay is assessed and described. METHODS: Chinese hamster ovary cells expressing a chimeric thyrotropin receptor were treated with bovine (b) TSH and different concentrations of an immunoglobulin G (IgG) monoclonal human TBAb (K1-70). TBAb was measured as a function of luciferase activity relative to bTSH alone and expressed as percent inhibition. Results obtained in the chimeric cell line were compared with those of a wild-type cell line. Analytical performance studies were subsequently performed with the chimeric cell line only. RESULTS: Immunodepletion of K1-70 IgG by using a protein G-Sepharose column showed that positive percent inhibition in the TBAb bioassay was detectable from K1-70 IgG only. The limit of blank was determined to be 12.2%. The limit of detection was 14% inhibition, equivalent to 0.4 ng/mL K1-70, while the limit of quantitation was 22% (coefficient of variation [CV] 12%) equivalent to 0.625 ng/mL K1-70. The dynamic range was between 14 ± 3.7 (mean % inhibition ± standard deviation) and 101 ± 2.6, equivalent to 0.4-10 ng/mL K1-70. The linear range was between 22 ± 2.6 and 93 ± 0.6 inhibition, equivalent to 0.625-5 ng/mL K1-70. The upper limit of the 99th percent reference range was 34% inhibition. In two laboratories, CV values for the intra- and inter-assay precisions for K1-70 ranged from 2% to 12% and from 1.7% to 14.5%, respectively. For patient sera, the CV values for the intra- and inter-assay precisions ranged from 3% to 9% and from 3% to 11%, respectively. No interference was found when follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotrophin were tested in the TBAb bioassay. The median of % inhibition values in 40 TBAb positive sera from patients with autoimmune thyroid disease were 93.5 (range 25-103) and 92 (range 64-107) for the wild type and chimeric cell lines, respectively. Further, all 40 samples of patients with various non-thyroidal autoimmune diseases were TBAb negative. CONCLUSIONS: This TBAb bioassay exhibits excellent analytical performance and high level of reproducibility.
BACKGROUND AND OBJECTIVE: A cell-based bioassay for the measurement of thyroid blocking autoantibodies (TBAb) has been recently reported. The analytical performance and validation of this bioassay is assessed and described. METHODS:Chinese hamster ovary cells expressing a chimeric thyrotropin receptor were treated with bovine (b) TSH and different concentrations of an immunoglobulin G (IgG) monoclonal humanTBAb (K1-70). TBAb was measured as a function of luciferase activity relative to bTSH alone and expressed as percent inhibition. Results obtained in the chimeric cell line were compared with those of a wild-type cell line. Analytical performance studies were subsequently performed with the chimeric cell line only. RESULTS: Immunodepletion of K1-70 IgG by using a protein G-Sepharose column showed that positive percent inhibition in the TBAb bioassay was detectable from K1-70 IgG only. The limit of blank was determined to be 12.2%. The limit of detection was 14% inhibition, equivalent to 0.4 ng/mL K1-70, while the limit of quantitation was 22% (coefficient of variation [CV] 12%) equivalent to 0.625 ng/mL K1-70. The dynamic range was between 14 ± 3.7 (mean % inhibition ± standard deviation) and 101 ± 2.6, equivalent to 0.4-10 ng/mL K1-70. The linear range was between 22 ± 2.6 and 93 ± 0.6 inhibition, equivalent to 0.625-5 ng/mL K1-70. The upper limit of the 99th percent reference range was 34% inhibition. In two laboratories, CV values for the intra- and inter-assay precisions for K1-70 ranged from 2% to 12% and from 1.7% to 14.5%, respectively. For patient sera, the CV values for the intra- and inter-assay precisions ranged from 3% to 9% and from 3% to 11%, respectively. No interference was found when follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotrophin were tested in the TBAb bioassay. The median of % inhibition values in 40 TBAb positive sera from patients with autoimmune thyroid disease were 93.5 (range 25-103) and 92 (range 64-107) for the wild type and chimeric cell lines, respectively. Further, all 40 samples of patients with various non-thyroidal autoimmune diseases were TBAb negative. CONCLUSIONS: This TBAb bioassay exhibits excellent analytical performance and high level of reproducibility.
Authors: Tanja Diana; Andreas Daiber; Matthias Oelze; Susanne Neumann; Paul D Olivo; Michael Kanitz; Paul Stamm; George J Kahaly Journal: J Clin Endocrinol Metab Date: 2018-10-01 Impact factor: 5.958
Authors: Tanja Diana; Christian Wüster; Paul D Olivo; Angelica Unterrainer; Jochem König; Michael Kanitz; Artur Bossowski; Brigitte Decallonne; George J Kahaly Journal: Eur Thyroid J Date: 2017-08-08
Authors: Tanja Diana; Hans-Peter Holthoff; Julia Fassbender; Christian Wüster; Michael Kanitz; George J Kahaly; Martin Ungerer Journal: Eur Thyroid J Date: 2020-07-23
Authors: G J Kahaly; S Ignjatović; M Sarić Matutinović; T Diana; B Nedeljković Beleslin; J Ćirić; M Žarković Journal: J Endocrinol Invest Date: 2021-07-29 Impact factor: 4.256