A New Assay for Thyrotropin Receptor Autoantibodies

Abstract

A new procedure for measuring patient serum thyrotropin receptor (TSHR) autoantibodies is described in which the autoantibodies inhibit binding of a human monoclonal thyroid stimulating antibody M22 (labeled with biotin) to TSHR-coated enzyme-linked immunosorbent assay (ELISA) plate wells. In the assay, M22-biotin binding is detected by addition of streptavidin peroxidase. The M22 based assay was more sensitive than a similar ELISA based on inhibition of TSH-biotin binding to TSHR coated wells with 1 U/L of NIBSC 90/672 giving approximately 35% inhibition in the M22-based system compared to approximately 15% inhibition in the TSH-based ELISA. This had an important impact on the precision of the 2 assays with the M22-based ELISA showing an interassay coefficient of variation (CV) of 10% at 1 U/L whereas the TSH-based ELISA had an interassay CV of 20% at 1 U/L. Analysis of sera from 307 control subjects without a diagnosis of Graves' disease indicated that only 2 (0.65%) gave inhibition of M22 binding values of greater than 10% (11% and 12% inhibition). In the case of sera from 108 patients with Graves' disease (treated and untreated) 103 (95%) gave inhibition of M22 binding values of 14% or greater. Receiver operating characteristic (ROC) plot analysis showed that 100% specificity for TSHR autoantibody detection in Graves' disease was obtained at 95% sensitivity for the M22-based ELISA and 89% sensitivity for the TSH-biotin-based ELISA. Inhibition of M22 binding to the TSHR was closely correlated to inhibition of TSH binding in the 108 Graves' sera (r = 0.99). However, inhibition of M22 binding was almost always greater resulting in improved sensitivity and precision.

Relevance of TSH-receptor antibody levels in predicting disease course in Graves’ orbitopathy: comparison of the third-generation TBII assay and Mc4-TSI bioassay

“…Interestingly, the AUC of the thirdgeneration TBII assay was 0.817 and that of the Mc4-TSI assay was 0.868, which means that both of these TRAb assays have equivalently high power in predicting severe GO disease course. Smith et al 12 showed the higher sensitivity of the M22-based ELISA than the TSH-based ELISA in diagnosing GD. The human monoclonal TSAb (M22) 9,10 allows a third-generation TBII assay system where the TRAb inhibits binding of a labeled TSAb rather than labeled TSH to the TSH receptor.…”

“…However, the most newly developed thirdgeneration TBII assay inhibits binding of a labeled TSAb (monoclonal Ab clone #M22) rather than labeled TSH to the TSH receptor 9,10 This results in enhanced sensitivity and specificity vs earlier assays using radiolabeled TSH. 6,8,11,12 The TSI bioassay measures cyclic adenosine monophosphate production after TSAb binds to TSH receptor. [13][14][15] Thus, this method enables the identification of the functional characteristics of TRAb.…”

TSH-receptor autoimmunity in Graves' disease after therapy with anti-thyroid drugs, surgery, or radioiodine: a 5-year prospective randomized study

“…Assays used in clinical routine differ considerably in their ability to detect such antibodies (4,5), but recent generations of assays show high sensitivity and specificity (14,15). In the present study, TRAb was measured using a radioreceptor assay that was able to detect TRAb in 83% of the untreated patients.…”

“…In this method, 125 I-labeled bovine TSH compete with TRAb in serum samples to bind to purified porcine TSH receptors, followed by polyethylene glycol precipitation (13). After the completion of our study, new generations of TRAb assays have been developed (14,15), and it has been shown that a minor subset of sera from patients with Graves' disease are falsely negative using the assay employed in our study (15). For the present calculations and presentation of results, we included only patients who were TRAb-positive at the time of inclusion in the study and before therapy.…”

Accuracy of receptor-based methods for detection of thyrotropin-receptor autoantibodies: a new automated third-generation immunoassay shows higher analytical and clinical sensitivity for the differential diagnosis of hyperthyroidism

“…Recently, a new method for measuring TRAb has been described by Rees Smith [31], in which autoantibodies inhibit the binding of a human thyroid-stimulating mAb M22 [32] labelled with biotin to TSHR-coated ELISA plate wells. The method is called the manual third-generation TRAb assay.…”