Stimulating TSH receptor (TSHR) autoantibodies (sTRAb) cause hyperthyroidism (HT) in Graves' disease (GD). Current assays quantify the pool of all TSHR autoantibodies (Ab). We introduce the first in vitro assay for direct detection of sTRAb suitable for routine clinical diagnosis. A bridge assay is performed on microplates (MP) using recombinant chimeric hTSHR-rLH/CG receptors (chim-hTSHR) with epitopes only for stimulating autoantibodies. A C-term directed antibody anchors chim-hTSHR to MP, to which one arm of sTRAb binds. The second arm of the sTRAb bridges to chim-hTSHR fused with alkaline phosphatase. Applying chemiluminescent substrate sTRAb were quantified using a luminometer (CentroLIA LB 961). Chim-hTSHR stability allows assay performance at room temperature or 37°C. WHO standard for Thyroid-Stimulating Antibody 90/672, WHO 90/672 calibrated control, normal sera, and sera from GD patients (50µl) were assayed. Range of detection was established using WHO 90/672: between-run precision (lots and days) with CV <20% was shown from 0.3–50.0 IU/L at within-run CV <10%. The power of bridge assay to distinguish between sera positive or negative for sTRAb was excellent (ROC plot analysis). Values obtained for patient sera using bridge assay differ from corresponding values determined by commercial competitive TSHR autoantibody assay (TRAK®). Our novel in vitro assay for direct detection of sTRAb, due to double detection by two chim-hTSHR, demonstrates high statistical quality and clinical relevance. Value discrepancies between direct and indirect assay may be due to measurement of all TSHR Ab, i.e. blocking and stimulating, by bovine TSH displacement from hTSHR in TRAK® assay. Also TSH binds only partially to the epitopes of both autoantibody types. Similar to bioassay, bridge assay measures the sTRAb causing HT in GD but it is suitable for routine clinical diagnostic use, not time-consuming, inexpensive, and has potential for automation.
