Induction of TSH-receptor antibodies in patients with toxic multinodular goitre by radioiodine treatment.

Previous studies have indicated pre-existing subclinical Graves' disease (GD) in many patients with the scintigraphic diagnosis of disseminated thyroid autonomy (DISA) or toxic multinodular goitre (TMG) type A. After radioiodine (RAI) treatment, an increase or the induction of TSH-receptor antibodies (TRAbs) in patients with GD or TMG has been repeatedly reported.In the present study, we investigated whether RAI could induce TRAbs in patients with TMG in whom pre-existing GD was excluded with highly sensitive TBII and TSAB assays. Therefore, TRAbs, anti-thyroperoxidase antibodies (anti-TPO-Abs) and anti-thyroglobulin antibodies (anti-TG-Abs) were determined in 43 consecutive patients at the nuclear medicine outpatient clinic with the scintigraphic diagnosis of toxic adenoma (TA; n = 20) or TMG type A (n = 11) or type B (n = 12) before and after RAI treatment. After RAI therapy, we detected TRAbs in 36 % (4 of 11) of patients with TMG type A only, whereas TRAbs were not detectable in patients with TMG type B or in patients with TA. Furthermore, 3 of the 4 patients with detectable TRAbs after RAI showed positive anti-TPO-Abs before RAI therapy. These findings provide further evidence for pre-existing GD in patients with TMG type A or DISA as previously suggested. Therefore, patients with TMG type A and high anti-TPO-Abs seem to be at increased risk of developing TRAbs or side-effects such as relapse of hyperthyroidism or thyroid associated ophthalmopathy. These patients therefore require more frequent evaluation after RAI treatment.     

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.     

Prediction of remission or relapse for Graves' hyperthyroidism by the combined determination of stimulating, blocking and binding TSH-receptor antibodies after the withdrawal of antithyroid drug treatment.

The most likely reasons for the low predictive value of TSH-receptor antibodies (TRAbs) determinations in previous investigations are the biological heterogeneity of TRAbs and changes of the different stimulating (TSAb) or blocking (TSBAb) antibody bioactivities of TRAbs during the course of Graves' disease (GD), which have not been taken into account in most previous studies. Furthermore, in a recent study it has been demonstrated that the decline of TRAb values detected with highly sensitive hTBII or TSAB assays is not useful in evaluating remission or relapse of GD at the end of antithyroid drug treatment (ATDT). In order to make a thorough investigation of the predictive values of all different TRAb qualities for the recurrence for GD after the withdrawal, we investigated hTBII, TSAbs and TSBAbs in 54 consecutive patients with GD at the end of ATDT and 12 - 13.5 months after stopping ATDT. Using the TRAb values at the time of reinvestigation in a model, recurrence for GD was better predicted compared to the determination at the time of withdrawal of ATDT. Furthermore, using this model, the combined determination of hTBII, TSAbs, and TSBAbs revealed the highest level of significance for the prediction of remission or relapse of GD (OR = 15; p < 0.0001) compared to the detection of hTBII, TSAbs and TSBAbs alone. Therefore, significant changes of TSAbs after the end of ATDT and the biological heterogeneity of TRAb define the conditions for predicting remission or relapse of GD after ATDT by TRAb determinations. Consequently, our results suggest that the prediction of the individual course of GD can only be improved by combined determinations of all TRAb qualities (hTBII, TSAbs and TSBAbs) after the end of ATDT.     

Detecting TSH-receptor antibodies with the recombinant TBII assay: technical and clinical evaluation.

We evaluated the technical robustness of the new commercial TBII assay using human recombinant TSH-R, and describe its use for the clinician in the routine laboratory. The human recombinant TSH-R assay (DYNOtest TRAK human) was compared to a conventional TBII assay (TSH-REZAK). Specificity was adjusted at 99.1% for both assays by ROC plot analysis including 113 healthy individuals. Sensitivity in 115 patients with active Graves' Disease (GD) was 98.2% for the DYNOtest TRAK human compared to 68.4% for the TSH-REZAK (p<0.0001). Comparison of the ROC-calculated cut off confirmed the recommended cut-off for the DYNOtest TRAK human, since 11% inhibition of tracer equals 1 IU/L, which is recommended as the grey zone. At the recommended cut-off (2 IU/L, 22% inhibition), the sensitivity is still 93.9% with 100% specificity. The ROC plot-derived cut-off of the TSH-REZAK (4.4%, 2 to 10 U/L) is below the grey zone of 10-15 U/L. At the recommended cut off of 15 U/L, the sensitivity is 43.0% with a specificity of 100%. Both assays showed a good correlation (r = 0.82, p < 0.0001); however, assay comparison revealed a constant bias in favour of the DYNOtest TRAK human. Applying the ROC plot-derived cut-off of 11 % inhibition (1 IU/L) for the DYNOtest TRAK human, we found 15 of 50 patients with autoimmune thyroiditis (AIT) and 6 of 23 patients with goitre (all < 1.5 IU/L). These patients would have been missed using the recommended 2 IU/L. The difference in sensitivity between the DYNOtest TRAK human and the TSH-REZAK was highly significant in the GD group, but not in other groups, indicating that the DYNOtest TRAK human has a higher sensitivity for GD without compromising specificity. In summary, the proposed high sensitivity of the new TBII assay using human recombinant TSH-R could be confirmed with the commercial product. This method offers a clear advantage over conventional TBII assays to confirm or exclude the diagnosis of GD. The recommended cut-off is very stringent, and until we have more information on the clinical relevance of low-level TBII between 1 and 1.5 IU/L, those patients should be monitored for the development of autoimmune thyroid disease.     

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.     

Similarity and dissimilarity between clinical and laboratory findings, especially anti-thyrotropin receptor antibody in ophthalmic Graves' disease without persistent hyperthyroidism and hyperthyroid Graves' disease

The aim of this study was to investigate thyroid states, significance of anti-TSH receptor antibodies and the clinical courses of patients with euthyroid Graves' ophthalmopathy. The clinical and laboratory finding of 30 patients with euthyroid Graves' ophthalmopathy were briefly as follows: 1) normal sized thyroid or small goiter; 2) negative or weakly positive thyrotropin binding inhibitor immunoglobulin (TBII); 3) normal thyroid [99 m-Tc] pertechnetate uptake; and 4) frequent observations of low serum TSH values. Besides TBII, thyroid stimulating antibody (TSAb) was measured under low salt and isotonic conditions using FRTL-5 rat thyroid cells. Both TBII and TSAb titers were lower in euthyroid Graves' ophthalmopathy than in hyperthyroid Graves' disease. Serum TSH levels frequently became low in patients considered as euthyroid upon the first examination as well as in Graves' patients in remission, reflecting preceding or mild hyperthyroidism. In follow-up studies, these patients with mildly elevated thyroid hormone levels and low TSH levels seldom reached a state of persistent hyperthyroidism, when TBII was negative or only weakly positive.     

Relationship among thyrotropin (TSH), thyroid stimulating immunoglobulins, and results of triiodothyronine (T3) suppression test in patients with Graves' disease

To investigate the relationship between TSH and abnormal thyroid stimulator(s) in patients with hyperthyroid Graves' disease in whom normal thyroid hormone levels in the serum were maintained by antithyroid drug therapy and in patients with euthyroid Graves' disease, determinations were made of the TSH concentration, action of thyroid stimulating immunoglobulins (TSAb and TBII), and T3 suppression. Out of thirty-three patients with hyperthyroid Graves' disease, twelve patients with subnormal TSH levels were all non-suppressible according to the T3 suppression test results and the detectability of TSAb and/or TBII was as high as 75%. In three out of five patients with euthyroid Graves' disease, the serum TSH level was subnormal. All three showed non-suppressibility in the T3 suppression test and positive action of either TSAb or TBII. One of them became clinically thyrotoxic when the TSAb activity was further increased and TBII became positive, and was therefore diagnosed as having hyperthyroid Graves' disease. The present findings suggest that there are still abnormal thyroid stimulator(s) in patients with hyperthyroid Graves' disease who have low TSH, even if their thyroid hormone concentrations remain normal. Moreover, it is likely that some of the patients with euthyroid Graves' disease are actually in a state of subclinical hyperthyroidism because of the presence of abnormal thyroid stimulator(s).     

Evaluation of the ratio of T3 release stimulating antibodies to TSH-binding inhibiting antibodies during the course of Graves' disease

The mechanisms leading to a remission of Graves' hyperthyroidism are still unknown. One possibility would be that autoantibodies raised during the course of disease could change the composition of the autoantibody spectrum in such a way to counterbalance the action of stimulatory autoantibodies, thereby resulting in an induction of remission. Therefore, in the present study using a rigorous methodological approach we have characterized the portion of T3 release stimulating autoantibodies among the total body of TSH receptor antibodies, i.e. the TSAb/TBII ratio, over the course of a 12 month antithyroid therapy in 25 patients with Graves' hyperthyroidism. Further, we have evaluated the relation of the alteration of the antibody spectrum to the course of disease. The TSAb/TBII ratio was indeed found to be subject to considerable changes. The observed shift in the antibody composition was more often in favor of a relative increase in stimulatory inactive TBII. Nevertheless, the clinical course of patients showing a persistence of TBII despite the decline or even absence of TSAb proved to be variable. In conclusion, our data indicate that the spectrum of autoantibodies may change over the course of antithyroid therapy owing mostly to a relative rise in stimulatory less active autoantibodies. This phenomenon, however, is apparently not closely related to the course of disease.     

Site-directed mutagenesis of a portion of the extracellular domain of the rat thyrotropin receptor important in autoimmune thyroid disease and nonhomologous with gonadotropin receptors. Relationship of functional and immunogenic domains.

Residues 287 to 404 of the rat thyrotropin (TSH) receptor exhibit little homology to gonadotropin receptors. A large segment of this region, residues 303-382, has no determinants important for TSH to bind or elevate cAMP levels nor for the activity of thyroid-stimulating autoantibodies (TSAbs) from the sera of Graves' patients, i.e. deletions, substitutions, or mutations in this segment do not result in a loss of any of these activities in transfected Cos-7 cells. Critical residues for these activities do, however, flank both sides of this segment. Of particular interest, deletion or mutation of residues 299-301 and 387-395 results in a marked decrease in high affinity TSH binding but preserves the ability of a TSAb to increase cAMP levels. Tyrosine 385 is also of particular interest since its mutation to phenylalanine, alanine, threonine, or glutamine results in a receptor with a 20-fold decrease in the ability of TSH to bind or increase cAMP levels, but one whose TSAb activity is, once again, preserved. Because one activity is preserved, we can conclude that (a) the receptor must be fully integrated within the membrane of the cell without malfolding, (b) these sequences represent determinants involved in the high affinity TSH binding site, and (c) separate determinants exist for high affinity TSH binding and TSAb activity, consistent with the existence of autoantibodies in Graves' sera which inhibit TSH binding (TBIAbs) or which increase cAMP levels (TSAbs). Additionally, we show that a 16-mer peptide (residues 352-367), which reacts with the sera of greater than 80% of patients with Graves' disease, can induce the formation of antibodies to a peptide with no sequence homology, residues 377-397. This peptide flanks the region, residues 303-382, with no determinants important for TSH receptor binding or activity. As noted above, it contains residues involved in the high affinity TSH binding site but whose deletion or mutation has no effect on TSAb activity, i.e. residues which would appear to be required at an epitope important for TBIAb but not TSAb antibody activity.     

Immunoglobulins from Graves' disease patients interact with different sites on TSH receptor/LH-CG receptor chimeras than either TSH or immunoglobulins from idiopathic myxedema patients

To examine the identity of binding sites for thyrotropin (TSH) and thyroid stimulating antibodies (TSAbs) associated with Graves' disease, we constructed eight human TSH receptor/rat LH-CG receptor chimeras. Substitution of amino acid residues 8-165 of the TSH receptor with the corresponding LH-CG receptor segment (Mc1 + 2) results in a chimera which retains high affinity TSH binding and the cAMP response to TSH but loses both the cAMP response to Graves' IgG and Graves' IgG inhibition of TSH binding. Two of three IgGs from idiopathic myxedema patients which contain thyroid stimulation blocking antibodies (TSBAbs) still, however, react with this chimera. Chimeras which substitute residues 90-165 (Mc2) and 261-370 (Mc4) retain the ability to interact with TSH, Graves' IgG, and idiopathic myxedema IgG. The data thus suggest that residues 8-165 contain an epitope specific for TSAbs and that TSH receptor determinants important for the activities of TSAbs and TSH are not identical. Further, binding sites for TSBAbs in idiopathic myxedema may be different from receptor binding sites for both Graves' IgG TSAb as well as TSH and may be different in individual patients.     

Interaction of thyroid-stimulating antibody with Graves' thyroid-stimulating hormone-binding antibody

OBJECTIVE: Evidence of anti-thyroid-stimulating hormone (TSH) antibody in Graves' serum has been reported. We found that extremely high Graves' anti-TSH antibodies neutralized other Graves' thyroid-stimulating antibody (TSAb) activity. METHOD: TSAb-IgG was affinity-purified by Sepharose-bound Graves' anti-TSH antibody (extremely high). RESULT: The thyroid-stimulating activity in affinity-purified TSAb-IgG increased about 4-5 times compared to that before purification. TSH-binding inhibitory immunoglobulin (TBII) activity in affinity-purified TSAb-IgG also increased using TSH receptor-coated tube assay. A similar increase of thyroid-stimulating activity accompanied with TBII activity was also observed in affinity-purified TSAb-IgG-F(ab')(2). CONCLUSION: This suggests the possibility that either TSAb may be an anti-idiotypic antibody against anti-TSH antibody or anti-TSH antibody may be an anti-idiotypic antibody against anti-TSH receptor antibody.     

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.     

Lack of interaction in recombinant human FSH receptor and both TSAb and TSBAb

Since cross-reactivity of TSH with the human FSH receptor has been reported, in this study we tested the effect of thyroid-stimulating antibody (TSAb) and thyroid stimulation-blocking antibody (TSBAb) on Chinese hamster ovary cells expressing human FSH receptor (CHO-hFSH-R cells). We examined the TSBAb activity of sera from hypothyroid patients who had a positive TBII to determine whether these sera also block the effect of FSH on CHO-hFSH-R cells. Although human FSH I-3 (0.25-16 ng/ml) stimulated the production of intracellular cAMP in CHO-hFSH-R cells with dose-responsive manner, neither TSAb nor TSBAb had such an effect on the cells.     

Augmentative effect of polyethylene glycol, polyvinyl alcohol and dextran on thyroid-stimulating antibody stimulated cAMP production in FRTL-5 cells and CHO cells expressing human TSH receptor

Previously we reported the augmentative effect of nonionic hydrophilic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA) and dextran on thyroid-stimulating antibody (TSAb) activity in porcine thyroid cell assays. We examined whether a similar phenomenon occurs in FRTL-5 thyroid cells and CHO cells expressing the human (h) TSH receptor (CHO-hTSHR cells). As with porcine thyroid cells, PEG 22.5% precipitated crude IgG from serum of patients with Graves' disease, significantly increased cAMP production as compared with PEG 12.5% precipitated crude IgG in both FRTL-5 cells and CHO-hTSHR cells. PEG 5% augmented purified-TSAb-IgG-stimulated cAMP production in both cell assays. TSAb activities and positivity by the direct assay using whole serum (0.05 ml) in the presence of 5% PEG in untreated Graves' patients were significantly increased as compared with the absence of 5% PEG. The augmentative effects of PVA 10% or dextran T-70 10% on TSAb-IgG-stimulated cAMP production were also observed in both cell assays. PVA 10% did not augment TSH-stimulated cAMP production in spite of weak augmentation by dextran 10% in both cell assays. Lack of the augmentative effects of PEG 5%, PVA 10% and dextran 10% on cAMP produced by GTPgammaS, forskolin and pituitary adenylate cyclase activating polypeptide was observed in both cell assays. The augmentative effects of these polymers in both cell assays similar to porcine thyroid cells suggest that there is no apparent species specificity among human, porcine and rat thyroid cells as far as TSH receptor linked cAMP production in cell membranes existed.     

Rabbit antibodies against two different extracellular domains of human thyrotropin receptor possess thyroid stimulating activities

We have produced rabbit antibodies against synthetic peptides corresponding to the mid-region (amino acid residues 172-202, C peptide) and to the unique segment near the transmembrane region (amino acid residues 341-370, P peptide) in the extracellular component of the human thyrotropin (TSH) receptor and evaluated their biological activities. Both anti-C peptide antibodies raised in two rabbits showed strong thyroid stimulating activities (TSAb) (4127% and 2548%). Anti-P peptide antibodies raised in two rabbits were also strongly positive for TSAb activities (359% and 3468%). However, none of these antibodies had TSH-binding inhibitor immunoglobulin (TBII) activities. These results suggest that the domains responsible for TSAb are likely to span the entire extracellular component of the TSH receptor.     

Mutations in the mouse TSH receptor equivalent to human constitutively activating TSH receptor mutations also cause constitutive activity.

Constitutively activating mutations in the human thyroid-stimulating hormone (TSH) receptor (TSHr) have been identified as the most prevalent molecular cause of non-autoimmune hyperthyroidism. To investigate the feasibility of an animal model for non-autoimmune hyperthyroidism, we introduced two mutations in the mouse TSHr which had previously been identified in the human TSHr. The two human mutations showed strong differences in TSH binding, basal cAMP and IP accumulation. In the human TSHr, the Ile 486 Phe mutation causes a high increase of basal cAMP accumulation and also basal stimulation of the inositol phosphate pathway, whereas the Val 509 Ala mutation results in a low increase of basal cAMP accumulation without affecting IP signaling. RNA was isolated from mouse thyroid tissue and reverse transcribed. A 2.4 kb PCR product from the mouse TSHr was cloned into the pGEM-T vector system. Ile was substituted with Phe at codon 486 and Val with Ala at codon 509. These mutated mouse TSHrs were subcloned in the pSVL expression vector. After transient expression in COS-7 cells, basal and TSH-stimulated cAMP and IP accumulation, cell surface expression and TSH binding were determined and directly compared to the human TSHr. Whereas constitutively activating mutations of the human parathyroid hormone (PTH)/PTH-related peptide receptor showed little or no change in basal cAMP accumulation when introduced into the rat PTH/PTHrP receptor, these two mouse TSHr mutations resulted in constitutive activity similar to the homologous mutations in the human TSHr. Therefore, it should be possible to establish a mouse model for non-autoimmune hyperthyroidism by homologous recombination to study the pathogenetic mechanisms of non-autoimmune hyperthyroidism.     

Changes in thyroglobulin release-stimulating activity (Tg-RSA) in immunoglobulin G from patients with Graves' disease during therapy with thionamide drug

We have reported previously a new method for detecting thyroglobulin release stimulating activity (Tg-RSA) by human thyroid monolayer cells in IgGs from Graves' patients. We report here changes in Tg-RSA in sera of patients during treatment with thionamide drug. Nineteen untreated patients had their Tg-RSA, TSAb, TBII and serum Tg concentration (STg) followed up. Before treatment, Tg-RSA and TSAb were positive in all 19 patients. Three of them were TBII negative. During treatment with thionamide, of 16 patients who had positive Tg-RSA, TSAb and TBII before treatment, 6 patients continued so during the period of observation. Of the remaining 10 patients, 8 became TBII negative. TSAb only was found negative in one patient and Tg-RSA only declined to negative in another patient. Three patients whose TBII was initially negative, were observed to be negative in all three indicators after treatment. STg was higher than normal in all patients before therapy and changes in Tg-RSA in almost all patients were parallel with those in STg during treatment. From the observation during treatment with thionamide, our results suggest that Tg-RSA in Graves' patients appears to have similar properties to TSAb.     

Adipocyte-TSH-receptor-related antibodies in Graves' disease detected by immunoprecipitation

Fat cell TSH receptor-related antibodies were detected by immunoprecipitation of 125I-TSH-receptor complexes and the nature of the antibodies was analyzed. To 125I-TSH prebound to Triton-solubilized receptors from guinea pig fat tissues, 50 micrograms of immunoglobulin G (IgG) was added and precipitation was effected by the addition of antihuman IgG. Immunoprecipitation values in 13 patients with Graves' disease were significantly (p less than 0.05) higher than those in 11 normal subjects. No significant increase in the values was seen in 8 patients with Hashimoto's disease. No correlation was observed between immunoprecipitation values and titers of antimicrosomal and antithyroglobulin antibodies. Neither was there any correlation between the values and TSH-binding inhibitor immunoglobulins (TBII) detected by the radioreceptor assay. The IgG fractions positive for the immunoprecipitation antibody were found to be poor human thyroid stimulators (HTS) relative to their TBII activities. And a highly significant correlation was observed between TBII and HTS activities among IgGs without detectable antibody by immunoprecipitation (r=0.907; p less than 0.005; n=7). These findings 1) demonstrate that immunoprecipitation assay using fat cell TSH receptor may detect TSH receptor-related antibodies different from TBII in patients with Graves' disease and 2) suggest the antibodies may recognize determinants on the receptor or its vicinity that do not participate in the binding of TSH or thyroid stimulating antibody, and may interfere with thyroidal response to these stimulators.     

Development of chicken antibodies toward the human thyrotropin receptor peptides and their bioactivities.

We have synthesized four peptides (P2, P4, E3 and P1) corresponding to different segments of human thyrotropin (TSH) receptor. We have obtained antibodies by immunizing them to chickens, and antibodies are evaluated for their thyroid stimulating antibody (TSAb), thyroid stimulation blocking antibody (TSBAb) and TSH-binding inhibitor immunoglobulin (TBII) activities. None of the antibodies had TSAb activity. Antibodies against human TSH receptor specific region such as P2 and P4 (P2: No. 372-397, P4: No. 341-358) had TSBAb and TBII activities. Anti-E3 antibody (E3: the third putative extracellular loop, No. 649-661) had only TSBAb activity. Anti-P1 antibody (P1: high homology with pig LH/CG receptor, No. 398-417), however, had none. These results suggest that anti-TSH receptor antibodies to different antigenic epitopes show heterogeneity in their biological activities.     

A synthetic oligopeptide derived from human thyrotropin receptor sequence binds to Graves' immunoglobulin and inhibits thyroid stimulating antibody activity but lacks interactions with TSH.

An 11-residue oligopeptide, P-195, was synthesized to match human thyrotropin (TSH) receptor structure from No. 333 to 343 of amino acid sequence. Preincubation of 5 Graves' IgGs with P-195 up to 10 micrograms resulted in dose-dependent reductions of thyroid stimulating antibody (TSAb) activity. [125I] labeled P-195 was found to bind Graves' IgG. The bound radioactivity correlated significantly with their TSAb activity (N = 25, r = 0.587, p less than 0.01). A peptide having a completely reverse sequence as P-195 did not show such biological activity. The peptide did not affect TSH and thyrotropin binding inhibitor immunoglobulin (TBII) on their receptor binding nor biological activities. P-195 was concluded to have a part of TSAb binding sites.