TSH Receptor Autoantibodies Research Articles

Stimulating and Blocking Thyroid-Stimulating Hormone (TSH) Receptor Autoantibodies from Patients with Graves’ Disease and Autoimmune Hypothyroidism Have Very Similar Concentration, TSH Receptor Affinity, and Binding Sites

The distinct biological properties of TSH receptor (TSH-R) autoantibodies (TRAbs) from patients with Graves' disease (GD) are yet unexplained on the molecular level. Here we compare serum concentration, affinity to the TSH-R, and binding sites on the TSH-R of stimulating (TSAb) and blocking (TBAb) TRAbs. Four-step affinity purification using human recombinant TSH-R was performed with 22 TRAb-positive sera from GD patients (11 with only TSAb and 11 with only TBAb) and five control sera. Antibody concentration, TSH binding inhibition (TBII), and TSAb/TBAb activity of the purified TRAb were assessed. Labeled purified TRAbs were used for displacement studies with TRAb and an additional 30 patients and 10 control sera. TRAbs could be purified to 80-93% purity with recovery of the TBII and TSAb and TBAb activity. No TRAbs could be purified from healthy individuals. The mean +/- SD concentration of TRAb was 17.3 +/- 5.4 microg/IU for the TSAb sera (range, 9.6-25.9) and 18.2 +/- 8.5 microg/IU for the TBAb sera (range, 4.6-29.2), respectively (P = 0.79). Affinity was in the picomolar range for both TRAb subtypes with mean +/- sd dissociation constant of 167 +/- 109 pM (60-410 pM) for TSAb and 253 +/- 132 pM (80-410 pM) for TBAb (P = 0.12). Purified and labeled TSAb and TBAb showed a very similar binding pattern to the TSH-R in displacement studies with unlabeled TSAb/TBAb or unpurified patients sera, indicating binding sites on the TSH-R in close proximity to each other. TSAbs and TBAbs in the serum of patients with GD have similar characteristics. They are of low concentration with high affinity and have also similar binding epitopes on the TSH-R.

Effects of TSH Receptor Mutations on Binding and Biological Activity of Monoclonal Antibodies and TSH

The effects of an extensive series of mutations in the TSH receptor (TSHR) leucine-rich domain (LRD) on the ability of thyroid-stimulating monoclonal antibodies (TSMAbs) and TSH to bind to the receptor and stimulate cyclic AMP production in TSHR-transfected CHO cells has been investigated. In addition, the ability of a mouse monoclonal antibody with blocking (i.e., antagonist) activity (RSR-B2) to interact with mutated receptors has been studied. Several amino acids distributed along an extensive part of the concave surface of the LRD were found to be important for binding and stimulation by the thyroid-stimulating human MAb M22 but did not appear to be important for TSH binding and stimulation. Most of these amino acids important for M22 interactions were also found to be important for the stimulating activity of six different mouse TSMAbs and a hamster TSMAb. Furthermore, most of these same amino acids were important for stimulation by TSHR autoantibodies in a panel of sera from patients with Graves' disease. Amino acid R255 was the only residue found to be unimportant for TSH stimulation but critical for stimulation by all thyroid-stimulating antibodies tested (23 patient serum TSHR autoantibodies, M22, and all seven animal TSMAbs). About half the amino acids (all located in the N-terminal part of the LRD) found to be important for M22 activity were also important for the blocking activity of RSR-B2 and although the epitopes for the two MAbs overlap they are different. As the two MAbs have similar affinities, their epitope differences are probably responsible for their different activities. Overall our results indicate that different TSMAbs and different patient sera thyroid-stimulating autoantibodies interact with the same region of the TSHR, but there are subtle differences in the actual amino acids that make contact with the different stimulators.

Estimation of Serum TSH Receptor Autoantibody Concentration and Affinity

We have used the human monoclonal TSH receptor (TSHR) autoantibody (M22) as a labeled ligand in competition with individual patient TSHR autoantibodies (TRAb) to estimate their serum concentrations and affinities. TSHR coated tubes, (125)I-labeled M22 IgG and Fab, and patient sera IgG and Fab were used in these studies. In 15 patients with Graves' disease, TRAb concentrations ranged from 50 to 500 ng/mL of serum (5- 60 parts per million of total serum IgG) and TRAb IgG affinities from 3.0 +/- 1.0-6.7 +/- 1.54-10(10) L/mol (mean +/- SD; n=3). Fab fragment affinities were similar to those of intact IgG. Serum TRAb with blocking (TSH antagonist; 4 patients) activity had similar affinities (3.0 +/- 0.25-7.2 +/- 2.2-10(10) L/mol) to TRAb IgG from patients with Graves' disease, but blocking TRAb concentrations were higher (1.7 - 27 mg/mL of serum). The concentrations of TRAb that we observed in the sera of the 15 Graves' patient (0.33 - 3.3 nmol/L) can be compared with that of circulating TSH. In particular, a serum TSH concentration of 100mU/L (0.7 nmol/L) is in the same range as the concentrations of TRAb we observed. Such a TSH concentration (similar to that observed after injection of 0.9 mg of recombinant human TSH) would be expected to cause a similar degree of thyrotoxicosis as seen in Graves' disease. Consequently, the thyroid-stimulating potencies (i.e., activity per mol) of patient serum TRAb and human TSH appear to be of a similar magnitude in vivo as well as in vitro. Overall, our results indicate that serum TRAb affinities are high and show only limited variations between different sera whereas concentrations of the autoantibodies vary widely.

Effects of a Thyroid-Stimulating Human Monoclonal Autoantibody (M22) on Functional Activity of LH and FSH Receptors

The glycoprotein hormones luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyrotropin (TSH) show low-level cross-reactivity between their respective receptors (R). Patient serum autoantibodies to the thyrotropin receptor (TSHR) do not appear to cross-react with the luteinizing hormone receptor (LHR) or follicle-stimulating hormone receptor (FSHR), although the concentrations of autoantibody with which it is feasible to carry out experiments of this type are limited. Consequently, we have studied the effects of high doses of the thyroid-stimulating human monoclonal autoantibody (M22) on the LHR and FSHR. Chinese Hamster ovary (CHO) cells stably expressing the TSHR, LHR, and FSHR and purified M22 IgG preparations were used in the study. CHO-TSHR, CHO-LHR, and CHO-FSHR cells were incubated with bovine TSH (0.1-25mU/mL), human recombinant chorionic gonadotropin (hCG; 0.5-10mU/mL) or human recombinant FSH (100-5000mU/mL) or with M22 IgG (0.001-5.0 microg/mL), and the extracellular cyclic AMP was measured by radioimmunoassay. Cyclic AMP levels increased in a dose-dependent manner after incubation of CHO-TSHR cells with TSH or M22 IgG, and on a molar basis the effects of TSH and M22 were similar. Cyclic AMP stimulation was not detectable in CHO-LHR and CHO-FSHR cells after incubation with M22 IgG, whereas incubation with hCG or FSH, respectively, caused dose-dependent cyclic AMP stimulation. On a molar basis, concentrations of M22 IgG approximately 100x those of FSH causing clear stimulation were ineffective with CHO-FSHR cells. Similarly, molar concentration of M22 IgG 20,000x those of hCG causing clear stimulation had no effect on CHO-LHR cells. This study shows that at relatively high concentrations, M22 IgG is unable to stimulate cyclic AMP levels in CHO-LHR or CHO-FSHR cells, suggesting that TSHR autoantibodies have greater specificity for the TSHR than TSH itself.

Idiopathic central diabetes insipidus in children and young adults is commonly associated with vasopressin‐cell antibodies and markers of autoimmunity

Autoimmune targeting of hypothalamic-neurohypophyseal structures in children and young adults with posterior pituitary and anterior pituitary dysfunction, as well as pituitary stalk involvement, are not yet completely understood. We aimed to (1) evaluate the presence of circulating vasopressin-cell autoantibodies (AVPc-Abs) in young patients with central diabetes insipidus (CDI), (2) detect organ-specific autoantibodies as markers of autoimmunity, and (3) define the relationship between immune markers and neuroimaging findings. Twenty patients were evaluated at a median age of 16.3 years. Twelve patients had idiopathic CDI, six had Langerhans cell histiocytosis (LCH) and two had germinoma. AVPc-Abs were evaluated in 40 healthy children. Magnetic resonance imaging (MRI) of the hypothalamic-pituitary region was performed longitudinally in all subjects. Circulating arginine vasopressin (AVP), protein tyrosine phosphatase (IA2), glutamic acid decarboxylase (GAD), 21-hydroxylase (21-OH), endomysium antibodies (EMA), parietal cell (PCA), thyroid peroxidase (TPO), thyroglobulin (TG) and TSH-receptor (TSHr) autoantibodies were evaluated. Circulating AVPc-Abs were found in 15 patients (75%), nine with idiopathic CDI, four with LCH and two with germinoma; the pituitary stalk was involved in most of them. Five patients with idiopathic CDI showed a persistence of AVPc-Abs during follow-up and one became positive subsequently. Serum IA2 autoantibodies were demonstrated in 14 patients (70%) and 21-OH autoantibodies in three of them. In idiopathic CDI, circulating AVPc-Abs suggest an autoimmune involvement of the neurohypophyseal system. The identification of AVPc-Abs in subjects who could have either idiopathic CDI or LCH or germinoma, however, indicates that AVPc-Abs cannot be considered a completely reliable marker of autoimmune CDI. Thus, close clinical and MRI follow-up are needed because AVPc-Abs may mask germinoma or LCH.

Does Autoantibody-negative Graves’ Disease Exist? A Second Evaluation of the Clinical Diagnosis

Advanced technical methods are essential for accurate diagnosis of Graves' or Basedow's disease (GD). Inadequate methods may lead to a false diagnostic conclusion. We have analyzed the clinical features and methodology aspects of cases diagnosed as GD with negative findings for TSH receptor autoantibodies. The initial diagnosis was based on clinical findings (patient record, hypermetabolic state, goiter palpation) and laboratory testing (fT4 and TSH). From a total of 255 newly registered patients with GD, fifty-one (20%) were negative in a conventional porcine TBII assay. All fifty-one patients were retested with 131I or 99mTc uptake tests, thyroid scintigraphy, and a second-generation TBII assay. Results disclosed twenty-one cases (8.3%) with diagnosis other than GD: ten cases of autonomous hyperthyroidism (Plummer's disease), seven cases of painless thyroiditis and four cases of euthyroid endocrine ophthalmopathy. All twenty-one patients remained negative in the second-generation TBII assay. Measurement by second-generation TBII assay was performed on the remaining thirty patients initially found negative for TBII. As a result of this reevaluation, only 234 of the original 255 patients had GD. Of those, 231 (204 according to porcine plus 27 according to human TRAb assay) had detectable TBII (98.7%). This investigation stresses the problem of correct diagnosis and the methodological limitations in the assessment of laboratory parameter validity in GD. Based on this work, TSH receptor autoantibody-negative GD is extremely rare.

Contribution of Single Nucleotide Polymorphisms withinFCRL3andMAP3K7IP2to the Pathogenesis of Graves’ Disease

Recently six DNA variants, two of which (M55V and 001Msp) are present in nuclear factor-kappaB inhibitors SUMO-4 and MAP3K7IP2 and four of which (fcrl3_3, fcrl3_4, fcrl3_5, and fcrl3_6) modulate nuclear factor-kappaB binding and production of the B cell surface molecule FCRL3, have been reported to be associated with a number of autoimmune diseases. The aim of this study was to investigate the association of these polymorphisms with disease in a large UK Caucasian Graves' disease (GD) data set. The study was a case-control association study of six polymorphisms. The study was conducted at a UK academic department of medicine. Study population included 1056 GD patients and 864 controls. There were no interventions. Tests for association with disease were measured. No association with disease was found for the M55V single-nucleotide polymorphism (SNP). Association was, however, found between GD and the 001Msp SNP [odds ratio (OR) 1.19 (95% confidence interval [CI]) 1.03-1.37], fcrl3_3 SNP [OR 1.17 (95% CI 1.02-1.34)], fcrl3_5 SNP [OR 1.18 (95% CI 1.04-1.35)], and fcrl3_6 SNP [OR 1.20 (95% CI 1.05-1.36)]. The 001Msp SNP was found to be associated with the presence of TSH receptor autoantibodies [OR 1.75 (95% CI 1.09-2.79)]. Functional evidence suggests that the 001Msp, fcrl3_3, fcrl3_5, and fcrl3_6 SNPs could cause changes in B cell signaling and activation pathways that could account for their association with GD. Further replication in independent data sets and fine mapping of the surrounding gene regions are needed to confirm the magnitude of the effect and location of the etiological variant(s) present within these gene regions.

Binding, Stimulating and Blocking TSH Receptor Antibodies to the Thyrotropin Receptor as Predictors of Relapse of Graves’ Disease after Withdrawal of Antithyroid Treatment

TSH-receptor autoantibodies (TRAbs) are a valuable diagnostic tool for confirming a diagnosis of Graves' disease (GD). While there is evidence that high TRAb levels are associated with relapse of GD, whether a discrimination of TRAb into stimulating (TSAb) and blocking (TBAb) autoantibodies would benefit the clinician in terms of outcome prediction remains unclear. To address this issue, we have determined TRAb, TSAb and TBAb levels in serum samples of ninety-six euthyroid patients with GD taken four weeks after antithyroid drug withdrawal (ATDT). Forty-seven patients (49 %) underwent relapse of GD within two years. Amongst those, forty-one (87 %) had been positive for TRAb and thirty-five (74 %) for TSAb after treatment. All patients except one were negative for TBAb. The correlation between TRAb and TSAb in those treated GD patients was relatively weak (r = 0.268, p < 0.001). Based on a cut-off limit of 1.5 IU/l, the positive and negative predictive values with respect to prediction of relapse were too low for any clinical relevance (TRAb: 49 % and 54 %; TSAb: 51 % and 55 %). However, when a cut-off level above 10 IU/l was used, the positive and negative predictive values increased to 83 % and 62 %. The additional measurement of TSAb or TBAb in those samples after therapy did not add additional information, even at higher decision thresholds. In conclusion, differentiation of TRAb into TSAb and TBAb is of no help in the prediction of relapse of GD in euthyroid patients at the end of ATDT, and only high TRAb levels are associated with relapse.

Relevance of TSH Receptor Stimulating and Blocking Autoantibody Measurement for the Prediction of Relapse in Graves’ Disease

Recently, we demonstrated that higher levels of autoantibodies to the human TSH receptor (TBII) predict relapse of hyperthyroidism in Graves' disease (GD). The aim of this study was to extend this outcome prediction by dividing TBII into stimulating (TSAb) and blocking (TBAb) TSH receptor autoantibodies. Altogether, ninety patients (81 female, 9 male) were retrospectively analyzed; sixty-four patients (71 %) did not go into remission or relapsed, whereas twenty-six patients (29 %) went into remission (median follow-up: 17.5 months). TSAb and TBAb measurement was performed in a CHO cell bioassay with cAMP readout at the time of their first visit in our outpatient clinic (single point measurement in median 6.5 months after initial diagnosis). In the remission group, eighteen of twenty-six patients (69 %) were TSAb-positive, whereas fifty-three of sixty-four patients (83 %) were TSAb-positive in the relapse group (p = ns). The mean stimulation indices (SI) were 4.1 in the remission group and 12.9 in the relapse group, respectively (p = 0.015). By using a threshold of 10 SI, the specificity for relapse was 96.0 %, as only one in twenty patients with an SI above 10 went into remission during follow-up (PPV 95 %). Most TSAb-positive patients also had high levels of TBII. Neither group showed any difference with respect to blocking type autoantibodies, which were mostly negative in both groups. In summary, high TSAb levels are similar but not superior to TBII for predicting relapse in GD patients. In contrast, TBAb measurement does not add any valuable information in this context. In the clinical routine, TSAb/TBAb measurement may not play an important role for diagnosis or outcome prediction of GD, since sensitive 2 (nd) generation TBII assays are easier to perform and offer similar information to the clinician. Bioassays should be reserved for special clinical questions such as Graves' disease in pregnancy.

The significance of thyroid blood flow at the inferior thyroid artery as a predictor for early Graves’ disease relapse

We investigated the clinical usefulness of thyroid blood-flow measurement in predicting relapse of Graves' disease (GD) in comparison with known risk factors for GD relapse. Thyroid blood flow was measured in pulsed Doppler mode at the inferior thyroid artery (ITA), and the peak systolic velocity (PSV) calculated. ITA-PSV was measured in euthyroid GD patients (n = 79) immediately before withdrawal of anti-thyroid drug (ATD) and in healthy subjects (n = 17). In the 79 euthyroid GD patients, the values of free triiodothyronine (FT3), TSH receptor autoantibody (TRAb), ITA-PSV and thyroid volume were significantly higher in the relapse group (n = 40) than in the nonrelapse group (n = 39) and the Youden index of ITA-PSV was significantly higher than that of FT3, TSH, TRAb and vascular endothelial growth factor (VEGF). ITA-PSV may assist in the prediction of early GD relapse after ATD withdrawal.

Stimulating and blocking antibodies to the TSH-receptor have similar affinities and only subtle differences in their epitope recognition

TSH-receptor (TSH-R) autoantibodies (TRAb) in autoimmune thyroid disease have been known for their heterogeneous effect with either stimulation (TSAb) or blocking (TBAb) of their target, resulting in the clinical symptoms of hyperthyroidism or hypothyroidism. Over years these distinct properties were attributed to either different affinities or binding sites to the TSH-R. Aim of this study was the verification of this concept using for the first time both affinity purified TRAb from the serum of patients and monoclonal antibodies (moabs) to the TSH-R with TSAb or TBAb activity. Affinity purification of TRAb was performed from 11 patients with Graves' disease having strong TSAb activity and 11 hypothyroid autoimmune thyroid patients with strong TBAb activity. In parallel, murine moAbs were generated by genomic vaccination with TSH-R DNA, of which IRI-Sab2 and IRI-Sab3 showed particular high stimulating potency (in the nanomolar range), whilst 1H7 revealed strong blocking activity. After labelling and saturation studies on TSH-R the Kd of individual purified TRAb was between 0.6 and 4.4 10–10 M with no difference between TSAb and TBAb patients. A similar Kd in the 10–10 M range was seen for both the TSAb and TBAb moabs and also bovine TSH. Competition studies with TSAb sera from patients with Graves' disease (n=100), or having TBAb (n=8) or controls (n=104) for binding to the TSH-R revealed virtually identical patterns for all purified TRAb and all moabs, irrespective of their TSAb or TBAb properties. A subsequent mapping of the epitopes of the moabs revealed an overlap of the stimulating IRI-Sab2 and IRI-Sab3 with the blocking 1H7 at the concave horse shoe part of the TSH-R ectodomain, which is also the likely side of TSH binding. These two approaches challenge the notion that stimulating and blocking antibodies would recognize structurally distant epitopes on the TSH-R, or that they have different affinities. Their different clinical effects seem to be the result of subtle differences in aminoacid activation.

TSH-Receptor autoantibodies help to predict severity and outcome of Graves' ophthalmopathy

TSH-Receptor autoantibodies help to predict severity and outcome of Graves' ophthalmopathy

Relevance of TSH-Receptor stimulating and blocking autoantibody measurement for the prediction of relapse in Graves' disease

Recently, we demonstrated that higher levels of autoantibodies to the human TSH-Receptor (TRAK human) predict relapse of hyperthyroidism in Graves' disease (GD). The aim of this study was to extend this outcome prediction with the help of stimulating (TSAb) and blocking (TBAb) TSH receptor autoantibody measurement. Altogether, 89 patients (79 female, 10 male) were retrospectively analyzed, of whom 64 patients (72%) did not go into remission or relapsed, whereas 25 patients (28%) went into remission (median follow-up: 20,1 months). TSAb and TBAb measurement was performed in a CHO cell assay with cAMP readout at the time of their first visit in our outpatient clinic (single point measurement in median 4,4 months after initial diagnosis). Within the remission group 19/26 patients (73%) were TSAb positive, whereas in the relapse group 53/64 patients (83%) revealed TSAb positivity. The mean stimulation indices (SI) were 4,3 and 12,8 in the relapse and remission group, respectively (p=0,015). By using a threshold of 10 SI, the specificity for relapse was 96,0%, as only 1 of 20 patients with an SI above 10 went into remission during follow-up (PPV 95%). Most TSAb positive patients had also high levels of TBII. Both groups showed no difference with respect to blocking type autoantibodies. Most GD patients in both groups were negative for TBAb, and only 1/26 patients in the remission group and 6 of 64 patients who relapsed showed weak TBAb activity (between 30–40% inhibition). In summary, high TSAb levels (like high TBII levels) are useful for the prediction of relapse in GD patients. In contrast, TBAb measurement does not add any valuable information in this context. In the clinical routine TSAb/TBAb measurement does not play an important role for diagnosis or outcome prediction of GD, since the human TBII assay is easier to perform and offers similar, if not better information. Bioassays should be reserved for special clinical questions (e.g. Graves' disease in pregnancy).

Human Recombinant TSH Preceding a Therapeutic Dose of Radioiodine for Multinodular Goiters Has No Significant Effect in the Surge of TSH-Receptor and TPO Antibodies

Radioiodine (RAI) treatment has increasingly been used mostly in elderly patients with multinodular goiter (MNG) as an alternative for surgery. Recombinant human thyrotropin (rhTSH) has been demonstrated to increase the uptake of RAI and also to promote a more even distribution of radionuclide among the various nodules. We have compared the surge of autoantibodies to thyroid peroxidase (anti-TPO) and to the TSH receptor (TRAb) in two groups of patients with MNG. Group RAI (n = 15) received only RAI, and Group RAI+rhTSH (n = 15) received RAI 24 h after 0.45 mg of rhTSH intramuscularly. At baseline, all 30 patients had negative anti-TPO antibodies. After RAI, 16 patients (eight in each group) exhibited a positive anti-TPO test (range, 70-2359 U/mL). In the rhTSH-treated group, anti-TPO values were significantly higher (as compared to basal levels; p < 0.02) after 3 months of RAI treatment. After 12 months, the anti-TPO values decreased to lower but still positive concentrations in nine patients (Group RAI: three patients; Group RAI+rhTSH: five patients). Only one patient had a positive TRAb test at baseline (67.5% inhibition of the TSH binding). After RAI, positive TRAb values were present in 21/30 patients. After 6 months of RAI treatment, there was a significant increase of the TRAb values in Group RAI+rhTSH patients. After 12 months, only four patients had positive TRAb (Group RAI: three patients; Group RAI+rhTSH: one patient). Two patients, one of each group, had an elevation of free T4 levels and suppressed serum TSH values, indicating hyperthyroidism (Graves' disease). Bioassay of TSH receptor (TSHR) indicated absence of a significant elevation of cAMP in the medium before and after RAI treatment in all patients. Moreover, predominantly blocking TSHR autoantibodies were detected in six of the 30 patients (three of each group). Sera from these patients were able to reduce the TSH-stimulated cAMP generation by CHO cells. We conclude that the autoantibodies to the TSHR and to TPO may occur after RAI treatment of patients, either with or without previous stimulation by rhTSH. The antibodies to the TSH comprised a combination of agonist (stimulating) and antagonist (blocking) antibodies, which in most patients did not induce clinical and laboratory evidence of active Graves' disease.

Clinical Evaluation of 3rd Generation Assay for Thyrotropin Receptor Antibodies: The M22-biotin-based ELISA Initiated by Smith

Recently a new procedure for measuring serum TSH receptor (TSHR) autoantibody (TRAb) was reported by Smith et al. in which the autoantibodies inhibit binding of a human monoclonal thyroid stimulating antibody M22 (labeled with biotin) to TSHR-coated ELISA plate wells (pTRAb(3rd) assay). The aim of this study was to compare the performance of pTRAb(3rd) assay with pTRAb(2nd) assay based on inhibition of TSH-biotin binding to TSHR-coated ELISA plate wells. In addition, we evaluated the applicability of TRAb3rd assay to discriminate between untreated Graves' disease (GD) and painless thyroiditis (PT). Analysis of sera from 230 healthy controls indicated that only 1 (0.43%) gave inhibition of M22-binding values of greater than 15% (32.8% inhibition). To define the clinical cut-off point for a positive serum with autoantibodies to the TSHR, we performed receiver operating characteristic curve of the data from 244 untreated GD and three different control groups for pTRAb(3rd) assay. With a sensitivity of 99.6% at a cut-off of 14.5%, 22.0% and 22.0% inhibition of M22 binding, the specificity of healthy controls without PT, with PT and with PT excluding postpartum PT and PT during remission of GD was 99.6%, 96.6% and 97.5%, respectively. The pTRAb(3rd) assay was closely correlated to pTRAb(2nd) assay in the 244 untreated Graves' sera (r = 0.911). The pTRAb(3rd) assay detected 243 of 244 (99.6%) untreated GD, whereas 9.2% of PT and 6.7% of the subacute thyroiditis (SAT) were detectable. In contrast, pTRAb (2nd) assay detected 242 of 244 (99.2%) Graves' same sera, while 16.8 % from PT's same sera and 13.3% from SAT were detectable. In conclusion, pTRAb(3rd) assay has significantly (p = 0.0026) superior diagnostic accuracy for GD and PT, compared to that of pTRAb(2nd) assay.

A New Assay for Thyrotropin Receptor Autoantibodies

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.

Affinity-enrichment of thyrotropin receptor autoantibodies from Graves' patients and normal individuals provides insight into their properties and possible origin from natural antibodies.

We used purified recombinant TSH receptor (TSHR) antigen prepared in mammalian cells to affinity-enrich TSHR autoantibodies from Graves' patients' IgG. Autoantibody enrichment, assayed by TSH binding inhibitory activity, was 20- to 1000-fold. Thyroid-stimulating antibody activity enrichment, although more difficult to quantitate, was comparable. TSHR-autoantibody approximate affinities for the holoreceptor assessed indirectly by TSH binding inhibition were 4-27 x 10(-9) m, an underestimate because 100% TSHR autoantibody purity was not attained. Consistent with previous data for serum, highly enriched TSHR autoantibodies in three of four patients showed lambda light chain bias. However, in contrast to expectations, antigen-enriched IgG was skewed primarily toward IgG2 and IgG3, subclasses associated with polysaccharides and microorganisms, respectively. Subclass depletion studies on antigen-enriched IgG indicated that TSHR autoantibodies were predominantly IgG1 and, surprisingly, IgG4. As controls, we affinity-enriched pooled IgG from normal individuals on TSHR antigen. This enriched IgG had detectable TSH binding inhibitory activity, although with lower specific activity than, and lacking the thyroid stimulatory activity of, Graves' IgG. Moreover, these natural IgG class autoantibodies largely recognized the same conformational variation in the TSHR N-terminal region as disease-associated TSHR autoantibodies. These studies suggest that TSHR autoantibodies may arise from natural autoantibodies, possibly by class switching from cross-reacting antibodies to microorganisms.

Characteristics of a human monoclonal autoantibody to the thyrotropin receptor: sequence structure and function.

The properties of a human monoclonal antibody to the thyrotropin receptor (TSHR) (M22) with the characteristics of patient sera thyroid stimulating autoantibodies is described. Similar concentrations (pmol/L) of M22 Fab and porcine TSH had similar stimulating effects on cyclic adenosine monophosphate (cAMP) production in TSHR-transfected Chinese hamster ovary cells whereas higher doses of intact M22 immunoglobulin G (IgG) were required to cause the same level of stimulation. Patient sera containing TSHR autoantibodies with TSH antagonist (blocking) activity inhibited M22 Fab and IgG stimulation in a similar way to their ability to block TSH stimulation. Thyroid-stimulating monoclonal antibodies (TSmAbs) produced in mice inhibited 125I-TSH binding and 125I-M22 Fab binding to the TSHR but the mouse TSmAbs were less effective inhibitors than M22. These competition studies emphasized the close relationship between the binding sites on the TSHR for TSH, TSHR autoantibodies with TSH agonist activity, and TSHR autoantibodies with TSH antagonist activity. Recombinant M22 Fab could be produced in Escherichia coli and the recombinant and hybridoma produced Fabs were similarly active in terms of inhibition of TSH binding and cAMP stimulation. The crystal structure of M22 Fab was determined to 1.65 A resolution and is that of a standard Fab although the hypervariable region of the heavy chain protrudes further from the framework than the hypervariable region of the light chain. The M22 antigen binding site is rich in aromatic residues and its surface is dominated by acidic patches on one side and basic patches on the other in agreement with an important role for charge-charge interactions in the TSHR-autoantibody interaction.

P14.24: Prenatal diagnosis and management of fetal goiter associated with maternal Graves' disease

Graves'disease is the most common cause of hyperthyroidism in pregnancy and may cause fetal and neonatal thyroid dysfunction. A case of maternal Graves'disease treated with propylthiouracil (PTU) is presented and prenatal detection and management of fetal goiter are discussed. A 30-year-old-woman, gravida 3, para 2, with Graves'disease was treated with 300 mg/day PTU. Maternal serum concentration of free thyroxine (FT4) was 0.9 ηg/dL (normal 0.6–1.54), thyroid-stimulating hormone (TSH) 2.28 mU/L (normal 0.5–4.2) and TSH-receptor autoantibody (TRAb) 14% (normal less than 12%). Fetal thyroid enlargement was noted by sonography at 25 weeks' gestation. Cordocentesis was performed to assess fetal thyroid function. The fetal serum FT4 was 0.9 ηg/dL (normal 2.3–2.7) and TSH was 98.65 mU/L (normal 8.7–10.5) indicating hypothyroidism. PTU was decreased to 150 mg/day and the fetus was treated with injection of L-thyroxine (250 µg) into amniotic fluid at 32 and 35 weeks' gestation. The patient remained euthyroid and serial ultrasound scans showed a shrinking of the fetal goiter. Spontaneous onset of labor occurred at 39 weeks' gestation. The male neonate weighed 2630 g with no discernible thyroid enlargement. The thyroid function tests at birth and on 16th day of life showed euthyroid status. Maternal antithyroid antibodies in patients with Graves'disease, known as THS-receptor autoantibodies (TRAb), can result in stimulation or inhibition of fetal thyroid gland. PTU treatment of Graves'disease in pregnancy may be associated with neonatal goitrous hypothyroidism whereas transplacental passage of stimulating immunoglobulin may cause fetal goiter with hyperthyroidism. Therefore, when fetal goiter is detected, cordocentesis should be offered to assess fetal thyroid function. In cases of fetal hypothyroidism, intra-amniotic injection of thyroxine has shown to be effective in raising serum levels of thyroxine, reducing those of TSH and decreasing the size of goiter.

P14.25: Perinatal outcome of prenatally diagnosed congenital cystic adenomatoid malformation

Graves’disease is the most common cause of hyperthyroidism in pregnancy and may cause fetal and neonatal thyroid dysfunction. A case of maternal Graves’disease treated with propylthiouracil (PTU) is presented and prenatal detection and management of fetal goiter are discussed. A 30-year-old-woman, gravida 3, para 2, with Graves’disease was treated with 300 mg/day PTU. Maternal serum concentration of free thyroxine (FT4) was 0.9 ηg/dL (normal 0.6–1.54), thyroidstimulating hormone (TSH) 2.28 mU/L (normal 0.5–4.2) and TSH-receptor autoantibody (TRAb) 14% (normal less than 12%). Fetal thyroid enlargement was noted by sonography at 25 weeks’ gestation. Cordocentesis was performed to assess fetal thyroid function. The fetal serum FT4 was 0.9 ηg/dL (normal 2.3–2.7) and TSH was 98.65 mU/L (normal 8.7–10.5) indicating hypothyroidism. PTU was decreased to 150 mg/day and the fetus was treated with injection of L-thyroxine (250 μg) into amniotic fluid at 32 and 35 weeks’ gestation. The patient remained euthyroid and serial ultrasound scans showed a shrinking of the fetal goiter. Spontaneous onset of labor occurred at 39 weeks’ gestation. The male neonate weighed 2630 g with no discernible thyroid enlargement. The thyroid function tests at birth and on 16th day of life showed euthyroid status. Maternal antithyroid antibodies in patients with Graves’disease, known as THS-receptor autoantibodies (TRAb), can result in stimulation or inhibition of fetal thyroid gland. PTU treatment of Graves’disease in pregnancy may be associated with neonatal goitrous hypothyroidism whereas transplacental passage of stimulating immunoglobulin may cause fetal goiter with hyperthyroidism. Therefore, when fetal goiter is detected, cordocentesis should be offered to assess fetal thyroid function. In cases of fetal hypothyroidism, intra-amniotic injection of thyroxine has shown to be effective in raising serum levels of thyroxine, reducing those of TSH and decreasing the size of goiter.