title: Standardization of a bioassay for thyrotropin receptor stimulating autoantibodies.
abstract: BACKGROUND
  Cell-based bioassays for functional thyroid stimulating autoantibodies (TSAb) are sensitive diagnostic tools. However, there is no bioassay available that is standardized with international reference material. We aimed to promote the standardization of the test results among laboratories that perform TSAb bioassays and calibrate TSAb levels against the second international standard (IS) 08/204 from the National Institute for Biological Standards and Control (NIBSC).
  
  
  METHODS
  Serum TSAb activity was measured with a FDA-cleared bioassay that utilizes CHO cells expressing a chimeric thyrotropin receptor (TSHR) and a c-AMP response-element-dependent luciferase. The IS was applied for calibration. TSAb results were reported as percentage of specimen-to-reference ratio (SRR%) and converted into mIU/L.
  
  
  RESULTS
  The IS dose-response curve was obtained using concentrations from 0.3125 to 200 mIU/L. Mean TSAb SRR%±standard deviation (SD) values for the IS concentrations 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, 40, 60, 80, 100, 120, 160, and 200 mIU/L were 63±4 (CV 6.3%), 63±4 (6.3), 67±2 (3.0), 76±6 (7.9), 91±8 (8), 134±8 (5.9), 201±13 (6.5), 294±12 (4.1), 336±10 (3.0), 348±8 (2.3), 360±14 (3.8), 371±15 (4.0), 381±9 (2.4), and 389±10 (2.6), respectively. A total of 127 dilution experiments were performed using 12 high TSAb-positive sera from patients with Graves' disease. When diluting TSAb-positive sera, IS concentrations within the linear range 5, 10, 20, 40, and 80 mIU/L were used for the calibration curve. All standard curves had R(2) values >0.95. Low coefficient of variation (CV %) values for the IS calibration curve (4-6%) were obtained. Compared to bovine TSH, no significant differences were noted using either a pool of healthy donors or a normal serum as reference controls. The average IU measured value for the assay cutoff (SRR 140%) corresponded to 9.54±1.68 mIU/L, and clinical application was shown in 60 Graves' patients.
  
  
  CONCLUSIONS
  The TSAb bioassay demonstrated excellent performance in terms of linear range, limit of quantitation, and imprecision. The dilution experiments showed a high correlation coefficient and excellent reproducibility. Thus, TSAb levels can be reliably converted from SRR% to IU/L. These results offer the perspective of standardizing TSAb levels among laboratories and enable more accurate comparison of TSAb studies.
authors.affiliation: 1 Molecular Thyroid Research Laboratory, Johannes Gutenberg University Medical Center , Mainz, Germany .
authors.forename: Tanja
authors.initials: T
authors.lastname: Diana
authors.forename: Michael
authors.initials: M
authors.lastname: Kanitz
authors.forename: Markus
authors.initials: M
authors.lastname: Lehmann
authors.forename: Yunsheng
authors.initials: Y
authors.lastname: Li
authors.forename: Paul D
authors.initials: PD
authors.lastname: Olivo
authors.forename: George J
authors.initials: GJ
authors.lastname: Kahaly
mesh_terms: D000818:Animals; D001323:Autoantibodies / Q000032:analysis*; D001681:Biological Assay / Q000592:standards*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006111:Graves Disease / Q000276:immunology; D006801:Humans; D018828:Immunoglobulins, Thyroid-Stimulating / Q000032:analysis*; D011989:Receptors, Thyrotropin / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D001323:Autoantibodies; D018828:Immunoglobulins, Thyroid-Stimulating; D011989:Receptors, Thyrotropin