Variability among TSH Measurements Can Be Reduced by Combining a Glycoengineered Calibrator to Epitope-Defined Immunoassays

Abstract

Objectives: Measuring protein markers with variable glycosylation, such as thyroid-stimulating hormone (TSH), with high accuracy is not an easy task. Despite highly sensitive third-generation tests, discrepancies among TSH assays still remain unsolved and are the focus of important standardization efforts. Earlier work from our group showed that a lack of similarity in epitope expression between standards and samples may account for discordant hormone measurements. In this study, we aimed at producing a glycoengineered TSH with serum-type glycosylation and compared its immunological behavior to that of the international standards. Study Design: Recombinant glycoengineered TSH (rgTSH) was produced in glycoengineered Chinese hamster ovary cells to express a highly sialylated TSH and tested in newly designed assays. Two groups of assays targeting defined epitopes were constructed and TSH levels were estimated in a panel of 84 clinical samples (2.1-22.4 mIU/l) based on the use of the current 3rd IS 81/565, the 1st IRP 94/674 and rgTSH calibrations. Results: Calibration based on rgTSH was found to significantly reduce the percentage difference means of assays compared to the pituitary standard. We also found that a switch from a mIU/l (3rd IS 81/565) to ng/l (rgTSH) basis can be established within the normal as well as in the mid to upper normal range of TSH levels. Of interest, TSH assays targeting the main immunogenic region displayed variable TSH values, indicating that, in this region, epitopes should be defined for assays to deliver similar values. Conclusions: A glycoengineered TSH with serum-type glycosylation proved to be a new calibrator efficient in harmonizing TSH values.

New Immunoassays of Bioactive TSH Improve Detection of Thyroid Disorders

“…Highly sialylated recombinant TSH however proved to display immunological identity with serum TSH, especially from hypothyroid patients [17]. Recently, we could demonstrate that combining a defined selection of TSH epitopes to a calibration based on glycoengineered TSH is able to abolish discrepancies among assays and achieve harmonization of TSH values as now requested by regulatory bodies [28].…”

“…Stably triply transfected clones have been selected and rgTSH producedin a chemically defined medium (Life Technologies).Mass estimation of rgTSH was carried out by Amino Acid Analysis (Alphalyse) of a purified recombinant preparation followed by immunological assessment. rgTSH bioactivity has been measured in CHO cells expressing the recombinant human TSH-receptor and was found 3-fold more active than a commercial preparation in stimulating cAMP production [28].…”

“…We recently published on several new assays measuring bioactive rgTSH and delivering harmonized values [28]. 4 out of 7 of them have been evaluated in this study.…”

“…They were all compared to a commercial assay of IRMA generation used to harmonize all the samples collected from various hospitals. These assays were calibrated with rgTSH and designated as A to D: they all bind the same antigenic clusters in TSH although through distinct epitopes [28].To define the upper limit of the reference range for each of the 5 assays, we calculated the 97.5 th centile Table 1) [32]. We then analyzed the clinical and biological parameters of individuals diagnosed with elevated TSH for each assay, i.e.…”

“…In earlier studies, we have investigated various calibrators of TSH assays [17] including the use of a recombinant glycoengineered calibrator to measure bioactive TSH [28]. To achieve similar immunological behavior between this preparation and serum TSH, we designed new sandwich assays based on defined selection of epitopes.…”