Untreated Hyperthyroid Graves Research Articles

Clinical experience with a radioreceptor assay for TSH-binding inhibiting immunoglobulins (TBII).

The aim of the present study was to evaluate the clinical value of a commercial kit for determination of TBII. Forty-seven of 50 patients with untreated hyperthyroid Graves' disease were TBII positive (sensitivity 94%). TBII was in the normal range in all normal volunteers and in patients with simple goiter, thyroid cancer and in most cases of nonimmunogenic hyperthyroidism (19 of 22). After 12 months antithyroid drug therapy with methimazole of 21 patients the prevalence of positive TBII findings was 28%. In contrast to this, 50 percent of the patients had increased microsomal antibodies at the end of therapy. The determination of TBII by TRAK-assay proved to be a sensitive, specific and practical method. The assay can be used to differentiate between hyperthyroidism of autoimmune or nonimmunogenic origin. Even so this method seems to be helpful for the follow-up during medical treatment of patients with Graves' disease. The results indicate that persistence of increased TBII levels are markers of active Graves' disease and suggest that in this situation ablative measures should be performed. Normalization of TBII on the end of a longstanding antithyroid therapy does not exclude the possibility of relapse in the further course.

Sensitivity of Thyroids to Endogenous Thyrotrophin in Patients with Graves' Disease

T3 nonsuppressibility and TSH nonresponsiveness to TRH are characteristics of untreated hyperthyroid Graves' disease. Although the tests are commonly restored to normal during antithyroidal drug therapy, dissociation of TRH and T3 suppression tests have been observed in euthyroid Graves' disease and during drug therapy. Abnormalities of pituitary-thyroidal regulation and persistence of thyroid autoimmune disease have been found in Graves' patients following various modalities of therapy. The present study investigated in vivo sensitivity to endogenous TSH of thyroids in 144 Graves' disease patients following treatment with subtotal thyroidectomy, 131I, or antithyroidal drugs. After administration of TRH, thyroidal sensitivity to TSH was determined by the following: delta T3 (peak T3 minus basal T3), % increase in T3 (peak over basal), and the ratio of delta T3 to delta TSH. Significant differences in sensitivity to TSH in T3 nonsuppressible patients were found compared to suppressible patients regardless of their TSH responses to TRH or modality of therapy. These data suggest that measurements of serum T3 and TSH following TRH administration to determine in vivo thyroidal sensitivity may be substituted for the T3 suppression test as a confirmatory test for Graves' disease or as a prognosticator of relapse following therapy.

Relationships Existing Between the Serum Cytokine Levels and Bone Mineral Density in Women in the Premenopausal Period Affected by Graves' Disease with Subclinical Hyperthyroidism

We examined the relationships existing between serum cytokine levels and bone mineral density (BMD) in women of premenopausal age affected by Graves' disease with subclinical hyperthyroidism. The study population consisted of 21 women with untreated hyperthyroid Graves' disease (group H) (age, 36 ± 2 years), eight women with untreated subclinical hyperthyroid status (group SH) (age, 33 ± 5 years) and 10 healthy women (group N) (age, 35 ± 3 years). The following measurements were made in all patients: free T4 (f T4), free T3 (f T3), thyroid stimulating hormone (TSH), TSH receptor antibody (TRab), anti‐thyroid peroxidase antibody (anti‐TPO), anti‐thyroglobulin antibody (anti‐Tg), interleukin‐2 receptor (IL‐2r), interleukin‐4 (IL‐4), interleukin‐8 (IL‐8) and interleukin‐13 (IL‐13). IL‐2r and IL‐8 levels significantly increased in group H compared with group SH ( p < 0.01 and p = 0.05, respectively) and group N ( p < 0.001 and p = 0.02, respectively). IL‐4 and IL‐13 levels tended to be lower in groups H and SH compared with group N, although this difference did not reach statistical significance. Bone mineral density was significantly reduced in only two areas of the femur in group H compared with group N. There was no difference in BMD between groups SH and N. There was no correlation between thyroid hormones, serum cytokine levels and BMD in either group. In conclusion, these results suggest that there were no relationships existing between the serum level of these cytokines and BMD in women of premenopausal age affected by Graves' disease with subclinical hyperthyroidism.

The prevalence and clinical significance of blocking thyrotropin receptor antibodies in untreated hyperthyroid Graves' disease.

The goal of this study was to evaluate the clinical significance of the blocking thyrotropin receptor antibodies (TSHRAb) in Graves' disease. The amount of blocking and stimulating TSHRAb were measured in 200 patients with untreated hyperthyroid Graves' disease using several cell lines carrying different TSHR chimera. Stimulating TSHRAb were measured in Chinese hamster ovary (CHO) cells with wild-type human TSHR (CHO-hTSHR) or a TSHR chimera with residues 90-165 (Mc2) or 8-165 (Mc1+2) substituted by equivalent residues of rat luteinizing hormone/chorionic gonadotrophin (LH/CG) receptor or in FRTL-5 cells. Blocking TSHRAb were measured in Mc2 cells. The activities of different TSHRAb were assessed and clinical features were compared to patients who were positive or negative for blocking TSHRAb antibodies. Blocking TSHRAbs were detected in 18.5% of patients (37/200) with hyperthyroid Graves' disease. Patients with blocking antibodies had significantly lower mean stimulating TSHRAb activities than those without blocking antibodies in wild-type CHO-hTSHR cells (301 +/- 179 vs. 446% +/- 537%, p = 0.005). Mean stimulating TSHRAb activities measured by FRTL-5, Mc1+2, or Mc2 cells and mean thyrotropin receptor inhibitor immunoglobulin (TBII) activities were not different between the two groups. The patients with blocking antibodies were not different from those without blocking antibodies in age, gender ratio, initial serum free thyroxine (T4) levels, or goiter size. However, the prevalence of exophthalmos was higher (35.1% vs. 17.5%, p = 0.024) in the patients with blocking antibodies than those without. In summary, the presence of blocking TSHRAb is not rare in patients with hyperthyroid Graves' disease when measured with chimeric receptor expressing cells. Blocking TSHRAb in Graves' sera do not strongly antagonize the action of stimulating TSHRAb in vivo, but could be a major factor responsible for underestimation of stimulating TSHRAb activities measured by CHO-hTSHR. The association of blocking TSHRAb with ophthalmopathy suggests that the TSHRAb repertoire of Graves' patients is different in those who do and who do not have ophthalmopathy.

Clinical significance of a sensitive assay for thyroid-stimulating antibodies in Graves' disease using polyethylene glycol at high concentrations and porcine thyroid cells.

The Inui and Ochi group recently reported that cAMP production by porcine thyroid cells (PTC) was augmented more by polyethylene glycol (PEG) 22.5% precipitated fractions from almost all Graves' sera than those of PEG 12.5%. In the present study, thyroid stimulating immunoglobulin (TSI) activity was determined with PTC and prepared crude Ig fractions precipitated by two different concentrations of PEG (final concentrations 13.5% and 22.5%) from sera obtained from 117 Graves' patients. The activity of TSI determined by the PEG 13.5% assay and activity determined by the PEG 22.5% assay were designated as thyroid-stimulating antibody (TSAb) and sTSAb, respectively. At first we studied 55 TSAb-positive patients with untreated hyperthyroid Graves' disease and classified them according to the TSAb activity-below 500% (group 1) and above 500% (group 2). The positive stimulatory effect, arbitrarily defined as the ratio of sTSAb to TSAb, being more than 1.2, was observed in 85% of patients, and group 1 had a significantly (P<0.025) greater stimulatory effect (34/35, 97.1%) than group 2 (13/20, 65%). Subsequently, in 29 TSAb-negative patients, sTSAb was measured and detected in 26 (89.7%). Finally, sTSAb, TSAb and TBII were compared between patients presenting with recurrent Graves' disease and those with silent thyroiditis after withdrawal of antithyroid drug treatment for Graves' disease. sTSAb was detected in all 14 relapsed patients, but none of the 9 patients with silent thyroiditis had detectable sTSAb. In contrast, TSAb and TBII activities were found in only 7 (50.0%) of the 14 relapsed cases. The present paper demonstrated that the assay with a higher PEG concentration was found to be sensitive, specific and useful for the diagnosis and follow-up of Graves' disease after drug withdrawal, although the underlying mechanism remains unclear.

An elevation of stem cell factor in patients with hyperthyroid Graves' disease.

Graves' disease is an autoimmune disorder characterized by the presence of antibodies against thyrotropin receptor (TRAb). Stem cell factor (SCF), derived from bone marrow, is known to promote lymphohematopoiesis. To investigate the relation between the alteration in plasma levels of SCF, thyroid hormone status, and TRAb measured by thyrotropin binding inhibition (TBI), 13 untreated, 21 treated, and 4 relapsed hyperthyroid Graves' disease patients, 21 patients with Hashimoto's thyroiditis, 6 patients with subacute thyroiditis, and 11 control subjects were examined. In untreated hyperthyroid Graves' disease patients, serum levels of thyroxine (T4) and triiodothyronine decreased rapidly by methimazole treatment, and TBI decreased progressively, but variably. Simultaneously, the elevated plasma levels of SCF decreased gradually and progressively. The plasma levels of SCF correlated curvilinearly with the serum levels of T4. In 4 patients with relapsed hyperthyroid Graves' disease, TBI was marginally positive in 3 patients and negative in 1, but plasma levels of SCF were elevated significantly in all 4 patients. In patients with subacute thyroiditis and Hashimoto's thyroiditis with or without T4 replacement, plasma levels of SCF did not differ from that of controls. These findings indicate that the elevation of plasma levels of SCF relates to the longstanding thyrotoxic state and that short-term thyrotoxicosis does not significantly affect plasma levels of SCF. It remains to be determined whether the elevation in plasma levels of SCF is induced by excess thyroid hormone, reflecting the hypermetabolic state, or whether the elevation of plasma levels of SCF contributes to stimulation of lymphocytes producing TRAb.

Effect of thyrotropin-releasing hormone on serum thyroid hormones: a study in the patients with untreated and treated Graves' disease and subacute thyroiditis.

In order to investigate the extrapituitary action of TRH on the thyroid, serum T3, T4, and TSH levels after im administration of TRH were analyzed in 63 patients with untreated hyperthyroid Graves' disease, in 60 euthyroid patients with treated Graves' disease, in 8 patients with subacute thyroiditis, and in 140 healthy subjects. TRH administration in the healthy subjects resulted in a significant increase in serum T3 and T4 levels after 2 h. However, in the patients with untreated hyperthyroid Graves' disease, a significant decrease in serum T3 and T4 levels with undetectable TSH was found 2 h after TRH administration. In the patients with subacute thyroiditis, serum T3 levels also significantly decreased after TRH administration. When a decrease in serum T3 and T4 levels after TRH administration in the patients with hyperthyroid Graves' disease was analyzed in terms of thyroid microsomal antibody and thyroglobulin antibody, a decrease in serum T3 and T4 levels was largest in patients with thyroid microsomal antibody and thyroglobulin antibody. In contrast, an increase in serum T3 and T4 levels in response to TRH in the euthyroid patients with Graves' disease was largest in patients without thyroid autoantibodies. It is concluded that TRH acts directly on the thyroid to suppress the thyroid hormone secreting activity in the absence of circulating TSH and that thyroid autoantibodies affect thyroidal response after TRH administration.

111In-DTPA-D-Phe1] octreotide scintigraphy in thyroidal and orbital Graves' disease: a parameter for disease activity?

Visualization of malignant lymphomas and granulomatous disease is possible by [111In-DTPA-D-Phe1]octreotide scintigraphy through binding of the radioligand to somatostatin receptors on activated leukocytes. Because thyroidal and orbital tissues are infiltrated by activated leukocytes in Graves' disease, a cross-sectional study to visualize disease activity with [111In-DTPA-D-Phe1]octreotide scintigraphy was performed. A correlation between thyroidal [111In-DTPA-D-Phe1]octreotide accumulation and free T4 (disease expression) and thyroid binding-inhibiting immunoglobulins (disease activity) is present in untreated hyperthyroid Graves' disease. There is also a correlation between orbital [111In-DTPA-D-Phe1]octreotide uptake and the clinical activity score (disease activity) and total eye score (disease expression), respectively, in Graves' orbitopathy. Visualization of thyroidal and orbital Graves' disease is feasible, but further investigation is necessary to establish the role of [111In-DTPA-D-Phe1]octreotide scintigraphy in representing disease activity and expression and in predicting therapeutical outcome.

The iodine perchlorate discharge test before and after one year of methimazole treatment of hyperthyroid Graves' disease.

We have recently reported that many euthyroid patients with a history of Graves' disease treated years earlier with methimazole (MMI) have a positive iodide (500 micrograms)-perchlorate discharge test (I-ClO4 test), suggesting a permanent thyroid iodide organification defect. We now report the results of the I-ClO4 test in patients with hyperthyroid Graves' disease before beginning a 1-yr course of MMI therapy and 40 days after MMI was discontinued. Twenty-nine patients (25 women and 4 men; mean age, 38 +/- 1.7 yr) with their first episode of hyperthyroid Graves' disease were studied. Before MMI therapy, I-ClO4 tests were carried out, and serum T4, T3, and TSH were measured to confirm the diagnosis of hyperthyroidism. A positive I-ClO4 test is defined as more than 15% 131I discharged from the thyroid 1 h after the administration of 1 g KClO4. Patients were then treated with 20 mg MMI for the first 2 months and variable doses thereafter for the next 10 months to maintain euthyroidism. Serum T4, T3, and TSH were measured monthly. Forty days after MMI was discontinued, I-ClO4 tests were repeated, and serum T4, T3, and TSH were measured every 2 months thereafter. Before MMI treatment, the I-ClO4 test was positive in 20 of 29 patients (69%) and negative in 9. The favorable responses (normal serum T4 and T3 values) to MMI therapy were similar in both groups. We have thus far studied 16 patients after MMI was discontinued and 9 of 12 patients (75%) with a negative I-ClO4 test after MMI therapy, and 1 of 4 patients (25%) with a positive test remained in remission for a mean of 7 months. We conclude that the I-ClO4 test is frequently positive in patients with untreated hyperthyroid Graves' disease, suggesting either an inability to organify the increased iodide concentrated by the hyperfunctioning gland or the concomitant presence of Hashimoto's thyroiditis, which almost always is associated with a positive I-ClO4 test. The former hypothesis is more likely, because many patients with a positive I-ClO4 test before MMI therapy had a negative test after MMI was discontinued.

Classification of Eye Changes of Graves' Disease

Classification of Eye Changes of Graves' Disease

Intrathyroidal Accumulation of T Cell Phenotypes in Autoimmune Thyroid Disease

In this study we have correlated peripheral T cell subset phenotypes with intrathyroidal lymphocyte accumulation in patients with autoimmune thyroid disease (Graves' and Hashimoto's disease). Our study utilized euthyroid family members for one of our control groups (n = 48) thus significantly limiting familial, but not disease-specific, influences on these T cell phenotypes. Our principal new observations were found only in patients with Graves' disease. As previously reported, there was a decrease in CD8+ (suppressor/cytotoxic) T cells in the peripheral blood of patients with untreated hyperthyroid Graves' disease (n = 27) (mean +/- SEM, 19 +/- 1.1% in patients compared with 25 +/- 1.2% in controls, p = 0.03), a finding not observed in treated, euthyroid Graves' disease patients or their relatives. However, the relative number of CD8+ T cells, assessed by CD4:CD8 ratios, was increased in the intrathyroidal T cell populations (n = 10), when compared to normal and patient peripheral blood. There were no consistent changes in total CD4+ (helper) T cells in the peripheral blood of patients with treated and untreated Graves' disease but a reduction in CD4+2H4+ (suppressor-inducer) T cells was seen in patients undergoing surgery for Graves' disease (13 +/- 6.9% compared with 39 +/- 3.4%). Again, however, this T cell subset was increased within the target organ of the same patients (41 +/- 5.9%). These data point to either a selective accumulation, or a specific "homing", of certain T cell subsets within the thyroid gland of patients with Graves' disease where T cell differentiation may be strongly influenced by antithyroid drug treatment and the local immune environment.

The Effect of Dithiotreitol on thyroid Peroxidase and Microsomal Antigen Epitopes Recognized by Auto and Monoclonal Antibodies

The effect of disulphide bridges reduction of the microsomal antigen (Mic-Ag) and thyroid peroxidase (TPO) by dithiotreitol (DTT) has been investigated. The reaction of all 67 tested sera from untreated hyperthyroid Graves' and from 22 Hashimoto's patients with high microsomal antibodies (aAb) titer was diminished by 90-95% by DTT, at pH 9.6. The remaining 5-10% of the activity was not destroyed by DTT. The residual Mic-Ag after DTT reduction was able to inhibit the binding of all 45 Graves' and 22 Hashimoto's tested aAb's to the native microsomal antigen by 100% at high concentration. Reaction of affinity purified TPO with two monoclonal antibodies (mAb) were diminished by 80% to 95% by DTT pretreatment, while the reaction of one mAb with TPO was only slightly affected. The reaction of TPO and Mic-Ag with rabbit polyclonal anti-TPO serum (rabbit a TPO) was diminished by 60% by DTT pretreatment. The immunological reactivity of TPO with aAb's was diminished by 65% after DTT pretreatment. The microsomal antigen-aAb's complex was not destroyed by DTT. Results presented in this paper suggest conformational epitope structure of the Mic-Ag recognized by aAb's in patients with thyroid autoimmune disease (AITD).

Thyroid-Stimulating Activity in Sera of Normal Pregnant Women

To evaluate the effect of a thyroid stimulator on thyroid function in the sera of normal pregnant women, we measured thyroid-stimulating activity (TSA) using a highly sensitive bioassay based on cAMP accumulation in cultured rat FRTL-5 thyroid cells. Serum was pretreated with 10% polyethylene glycol (PEG), and the supernatant (PEG-pretreated serum) was then used in the following studies. FRTL-5 cells were preincubated in 5H medium and incubated for 2 h with PEG pretreated serum, and cAMP was measured. All 11 patients with untreated hyperthyroid Graves' disease with strongly positive thyroid-stimulating antibody activity had normal TSA, because only 5.6% of their immunoglobulin G was recovered in the PEG-pretreated serum. In 32 normal pregnant women, 29 (91%) had positive TSA. Their TSA showed statistically significant positive correlations with serum hCG and free T4 levels, and a negative correlation with serum TSH levels. Moreover, when hCG was absorbed from sera by incubation with the solid phase anti-HCG monoclonal antibody, a significant positive correlation was observed between the rate of decrease in hCG and that in TSA. In conclusion, 1) TSA exists in the sera of normal pregnant women, which reflects hCG itself; and 2) thyroid glands of normal pregnant women may be stimulated by TSA to induce a slight suppression of TSH but not sufficient to induce overt hyperthyroidism.

Activity of Thyroid Stimulating Antibody and Thyroid Stimulation Blocking Antibody Determined by Radioiodine Uptake into FRTL-5 Cells.

To investigate the pathophysiology of patients with autoimmune thyroid diseases, we measured serum thyroid stimulating antibody (TSAb) activity and thyroid stimulation blocking antibody (TSBAb) activity by determining the radioiodine (125I) uptake into FRTL-5 cells. FRTL-5 cells were pre-incubated for seven days with 5H medium and then incubated for 48 hours with patients' crude IgG prepared by polyethylene glycol precipitation. In order to measure TSBAb, 10 microU/ml TSH was also added. 125I was added one hour before the end of the 48 hour incubation period. After the incubation, the medium was aspirated, and the radioactivity in the cells was counted. In patients with untreated hyperthyroid Graves' disease, TSAb was detectable in 18 of 20 patients, the detectability being 90%, and activity showed a statistically significant positive correlation with TSAb activity determined by c-AMP accumulation. Out of 41 patients with hypothyroidism, TSBAb determined by 125I uptake was positive in six cases, the detectability being 14.6%. The inhibition of 125I uptake by one of these six IgGs was suggested to be at the TSH receptor level because it inhibited TSH induced c-AMP accumulation and showed positive thyrotropin binding inhibitor immunoglobulin (TBI I) activity, but did not inhibit the forskolin- and (Bu)2cAMP-induced 125I uptake. Inhibition of another IgG was suggested at the post-receptor level because it did not inhibit TSH induced cAMP accumulation and showed negative TBI I activity, but inhibited forskolin- and (Bu)2cAMP-induced 125I uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical studies on abnormal thyroid stimulators in patients with Graves' disease. I. A sensitive assay for thyroid-stimulating antibodies using cultured porcine thyroid cells and polyethylene glycol precipitation of serum

The activities of thyroid-stimulating antibody (TSAb) in serum from patients with Graves' disease were measured by a sensitive assay, using cultured porcine thyroid cells and the precipitation from serum with polyethylene glycol (PEG), and the activities were compared with those of thyrotropin binding inhibitor immunoglobulin (TBII), measured by the commercial assay kit. Porcine thyroid cells after digestion were cultured for 15-18 hours with TSH of 1-10,000 microU/ml or the precipitations of sera from normal subjects and patients with Graves' disease or Hashimoto's thyroiditis, and then the cAMP levels in the culture medium were determined by the commercial RIA assay kit (Yamasa). The precipitation was obtained by adding 0.5 ml of 30% PEG solution to 0.5 ml serum, and was resuspended with 0.6 ml of Hanks' medium without NaCl, containing 1.5% bovine serum albumin, 20mM Hepes and 0.5 mM 3-isobutyl-1-methylxanthine. The precipitation contained about 85% of immunoglobulin and 63% of albumin of the original amount of the serum, as well as substantial TSH, when the original serum contained TSH more than 40 microU/ml. When the PEG precipitations from 10 normal subjects were incubated with the thyroid cells of 4 X 10(5) cells, the cAMP releases into the medium ranged from 83 to 124%, when the mean value was calculated as 100%. Therefore, the cAMP release of more than 130% of the amount released into the culture medium incubated with normal IgG was judged as positive TSAb activity. The minimum detectable quantities were regarded as about 5 microU/ml TSH equivalent. TSAb and TBII activities were detected in 48 (92%) and 50 (96%) of 52 patients with untreated hyperthyroid Graves' disease, respectively, and either TSAb or TBII activities were detected in 16 (80%) of 20 patients with Graves' disease maintained in a clinically euthyroid state by treatment with antithyroid drugs. TBII was positive in 10(50%) of these patients. Some patients showed distinct discrepancies in these two activities, although there was a significant positive correlation between TSAb and TBII activities (r = 0.53, p less than 0.01) in patients with untreated Graves' disease. In these patients, TSAb activities showed a significant positive correlation with values for 99mTc thyroid uptake, determined 30 min after the injection. However, they did not show any significant correlation with serum T4 or T3 concentrations. Similarly, TBII showed significant correlations with goiter size and 99mTc thyroid uptake. To conclude, the present assay for TSAb is sensitive and reproducible.(ABSTRACT TRUNCATED AT 400 WORDS)

Reduction in the suppressor-inducer T cell subset and increase in the helper T cell subset in thyroid tissue from patients with Graves' disease.

The expression of surface markers associated with activation and characterization was compared among T cells in thyroid glands and peripheral blood of 10 patients with Graves' hyperthyroidism receiving chronic antithyroid drug therapy, in peripheral blood of 15 patients with untreated hyperthyroid Graves' disease, and in peripheral blood of 21 normal subjects using two-color flow cytometry. In the chronically treated Graves' disease patients, the percentage of activated T cells (HLA-DR+ T cells) among total T cells was significantly higher in thyroid tissue than in peripheral blood, and the increase in percent activated T cells was also significant among both helper/inducer T cell (CD4+ cell) and suppressor/cytotoxic T cell (CD8+ cell) subsets. The percentage of activated T cells in peripheral blood was not significantly different between chronically treated hyperthyroid Graves' patients and normal subjects, whereas the percentage of activated T cells in the peripheral blood from untreated hyperthyroid Graves' disease patients was significantly higher than that in normal subjects or chronically treated hyperthyroid Graves' patients. The percentages of CD4+ cells and CD8+ cells among total T cells were not different between thyroid tissues and peripheral blood in patients with chronically treated hyperthyroid Graves' disease. When CD4+ were further divided into helper T cells (CD4+2H4- cells) and suppressor-inducer T cells (CD4+2H4+ cells) using two-color flow cytometry, the percentage of helper T cells among CD4+ cells was significantly higher in thyroid tissue than in peripheral blood, resulting in an increased ratio of CD4+2H4- cells to CD4+2H4+ cells. The percentage of CD4+2H4+ cells in peripheral blood, however, was not significantly different among untreated and chronically treated Graves' disease patients and normal subjects. From the findings of abnormalities in intrathyroidal T cell subsets, we suggest that the decrease in the function of suppressor T cells within the thyroids of Graves' disease patients may be due to a decrease in CD4+2H4+ cells within thyroid tissue.

Clinical usefulness of a highly sensitive enzyme-immunoassay of TSH.

Using a recently developed sensitive enzyme immunoassay (EIA) for TSH, serum TSH levels in normal subjects and patients with various thyroid disorders were measured. The minimum detectable concentration was less than 0.005 mU/l. An excellent correlation was observed between TSH values measured by EIA and by sensitive radioimmunoassay (r = 0.932). In 26 normal subjects, serum TSH ranged from 0.30 to 2.65 mU/l (geometric mean 0.97 mU/l). In 27 untreated hyperthyroid Graves' patients, serum TSH was detected in all but one, ranging from 0.005 to 0.19 mU/l (geometric mean 0.040). These values were proved to be specific for human TSH by absorption, recovery, cross-reactivity and dilution studies; non-specific serum effects were also ruled out. In 10 patients with destructive thyroiditis, similar TSH values (0.005-0.20 mU/l, mean 0.032) were observed. However, no significant correlations between TSH and circulating thyroid hormone levels were observed in these thyrotoxic conditions. Furthermore, 9 untreated Graves' patients did not respond to a single i.v. injection of TRH. In 13 hypothyroid patients with hypothalamo-pituitary disorders, 10 had basal TSH levels of less than 0.4 mU/l, and TRH tests in five gave peak TSH values of more than 0.8 mU/l, which were associated with significant increases in serum T3. In three patients with Sheehan's syndrome, elevated basal TSH levels were observed. Two of them responded to TRH, but these TSH elevations were not associated with T3 increases. In conclusion, this sensitive EIA could measure TSH levels in most patients with thyrotoxicosis and central hypothyroidism. However, the true significance of these measured values needs to be elucidated by future studies.

Enzyme-linked immunosorbent assay of autoantibodies reacting with thyroid plasma membrane antigens in sera of patients with autoimmune thyroid diseases.

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of autoantibodies reacting with thyroid plasma membrane antigens has been established. Autoantibodies reacting with thyroid plasma membrane antigens were detected by the ELISA in 95% of untreated hyperthyroid Graves', 68% of antithyroid drug-treated Graves' up to four months of the therapy, in 62% of Hashimoto's thyroiditis and in 8.9% of toxic nodular goitre. The ELISA was negative in 100% healthy blood donors, 100% non-toxic nodular goitre, in 12 patients with rheumatoid arthritis, 18 patients with scleroderma and 94% of patients with systemic lupus erythematosus. The mean value of autoantibodies titre was higher in untreated hyperthyroid Graves' (1:84,000) and lowest in positive patients with autoimmune disease of non-thyroid origin (1:4000). The cross-reactivity of antimicrosomal antigen antibodies was below 10%; there was no influence of antithyroglobulin antibodies on the ELISA; and most of the autoantibodies react with plasma membrane antigens different from the TSH binding sites.

A sensitive and practical assay for thyroid-stimulating antibodies using crude immunoglobulin fractions precipitated with polyethylene glycol.

A simple, sensitive, and practical assay for thyroid-stimulating autoantibodies (TSAb) was developed in which cryopreserved porcine thyroid cells were incubated with crude immunoglobulin fractions sedimented from serum with polyethylene glycol. In the assay, 1.4- to 2.0-fold and 6- to 12-fold increases in cAMP released into Hank's medium without NaCl were found at 1 and 10 microU/ml bovine TSH, respectively. TSAb were detected in 41 (97.6%) of 42 patients with untreated hyperthyroid Graves' disease, 29 (55.8%) of 52 patients with hyperthyroid Graves' disease who were euthyroid while taking antithyroid drugs, 22 (78.6%) of 28 patients with euthyroid Graves' disease, and none of the patients with simple goiter, adenomatous goiter, thyroid adenoma, or thyroid cancer tested. TSAb activities measured using porcine thyroid cells significantly correlated with those measured using human thyroid adenoma cells (r = 0.908; n = 46; P less than 0.001). Thyroid-stimulating activity was also detected in 11 (28.9%) of 38 patients with Hashimoto's thyroiditis. However, the activity was considered to be due to TSH in the patients' sera, because it was completely abolished by pretreatment with anti-TSH antibodies. Serum TSH concentrations lower than 50 microU/ml did not affect the assay result. In Graves' disease after cessation of antithyroid drugs, 85.7% (12 of 14) of TSAb-positive patients relapsed, while 77.8% (14 of 18) of TSAb-negative patients remained in remission. Thus, the assessment of TSAb was useful as an index to predict prognosis.

An enzyme-linked immunosorbent assay for the measurement of thyroglobulin in human serum using mouse monoclonal antibodies.

A robust, rapid, sensitive and specific enzyme-linked immunosorbent assay (ELISA), using an in-house immunoglobulin-A-sub-class mouse monoclonal human-thyroglobulin antibody (WNSM2), with a sensitivity of 1.51 pmol/l has been established for the measurement of thyroglobulin in serum. Standard curves in varying dilutions of human serum were similar to standard curves obtained in serum-free medium, thus demonstrating no significant cross-reactivity with any serum proteins other than thyroglobulin. Levels of serum thyroglobulin detected by ELISA correlated significantly (r = 0.93, P less than 0.001) with those from a standardized and well-characterized radioimmunoassay. The coefficients of variation within and between ELISA assays were 3.9 and 7.1% respectively. Thyroglobulin was detectable in 87% of 54 normal subjects who had no history of thyroid or autoimmune disease, the mean (+/- S.D.) for this group being 15 +/- 6.6 pmol/l with a range of 1.51-53 pmol/l. Using this assay, levels of thyroglobulin were shown to be significantly (P less than 0.002) increased in patients with untreated hyperthyroid Graves' disease compared with normal subjects.