Optimization and Clinical Assessment of a Radioreceptor Assay for Thyrotropin-Binding Inhibitor Immunoglobulins

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1 Endocrinol. Japon. 1987, 34 (1), Optimization and Clinical Assessment of a Radioreceptor Assay for Thyrotropin-Binding Inhibitor Immunoglobulins JUNJI KONISHI, KANJI KASAGI, YASUHIRO IIDA, TADAKO KOUSAKA, TAKASHI MISAKI, KEISUKE ARAI, YASUTAKA TOKUDA AND KANJI TORIZUKA Department of Nuclear Medicine, Kyoto University School of Medicine, Sakyo-ku, Kyoto606, Japan Abstract We tried to improve the sensitivity of a radioreceptor assay for thyrotropinbinding inhibitor immunoglobulins (TBII) by modifying assay conditions. About a twofold increase in sensitivity without a loss of reproducibility was obtained by prolonging the incubation of the receptors with test serum from15to120 min before the addition of125i-labeled thyrotropin. In20untreated, 49treated patients with Graves' disease and19patients with euthyroid Graves' disease, TBII activities obtained using120min preincubation were significantly higher than those obtained using15min preincubation (p<0.005). No significant increase in TBII activities was observed in the presence of sera from patients with primary hypothyroidism (n=17), simple goiter (n=7), adenomatous goiter (n=11), thyroid adenoma (n=11) or cancer (n=12). TBII were detectable in 15 (47%) of32triiodothyronine-nonsuppressible Graves' patients who were receiving maintenance antithyroid drug therapy using120min preincubation, while they were positive in only6patients (19%) using15min preincubation. The assay using a longer preincubation period was found to be sensitive, specific and useful for diagnosis and follow-up of Graves' disease. Since the development of the radioreceptor assay for thyrotropin (TSH), it has become evident that immunoglobulins which inhibit the binding of labeled TSH to the TSH receptor are present in sera of most patients with Graves' disease (Rees Smith and Hall, 1974) and in some with primary myxedema (Endo et al., 1978; Konishi et al., 1985). These TSH-binding inhibitor immunoglobulins (TBII) are now considered to be antibodies to the TSH receptor, and their etiological significance has been extensively studied (for review see Burman and Baker, 1985). Recent development of a radioreceptor assay using solubilized porcine thyroid membranes by Shewring and Rees Smith (1982) has made the detection of TBII easier and more reliable. They further simplified the assay by using unextracted serum directly (Southgate et al., 1984). In the present study we tried to improve the sensitivity of the assay by modifying the assay conditions and have evaluated the clinical usefulness of the improved assay. Received May12, 1986

2 14 KONISHI et al. Endocrinol. February1987 Japon. Materials and Methods Serum samples Serum samples were obtained from53normal subjects, 20patients with untreated Graves' disease, 49euthyroid patients with Graves' disease treated with a maintenance dose of antithyroid drugs (5-10mg/day methimazole or50-100mg/day propylthiouracil) for more than one year, 19 patients with euthyroid Graves' disease, 17 patients with primary hypothyroidism, 7 with simple goiter, 11with ademomatous goiter, 11 with thyroid adenoma and12with thyroid cancer. The diagnosis of Graves' disease was based on the presence of diffuse goiter, thyrotoxicosis and laboratory findings including elevated serum thyroxine, free thyroxine and triiodothyronine (T3) concentrations, elevated30min99mitc thyroidal uptake and detection of circulating antithyroid (thyroglobulin and microsomal) antibodies. Euthyroid Graves' disease was defined as ophthalmopathy of Graves' disease associated with thyroid autoimmunity such as diffuse goiter or:detection of antithyroid antibodies in euthyroid subjects. Patients with primary hypothyroidism comprised6patients with Hashimoto's thyroiditis diagnosed on the basis of histological findings obtained by needle biopsy and11nongoitrous patients who had elevated antithyroid antibody titers. The diagnosis of simple goiter was made on the basis of diffuse goiter and the histological findings obtained by needle biopsy. The diagnosis of ademomatous goiter, thyroid adenoma and thyroid cancer was based on the histology obtained at surgery. Commercially avialable radioimmunoassay kits (Dainabot Radioisotope Labs., Minato-ku, Tokyo105) were used to measure serum thyroxine and T3levels. Free thyroxine was measured with an Amerlex kit (Amersham International Ltd., Amersham, Buckinghamshire, HP7 9LL, U. K.). The serum TSH level was measured with a double antibody radioimmunoassay kit (Daiichi Radioisotope Labs., Chuo-ku, Tokyo, 103). Antibody titers to thyroglobulin and microsomes were also determined with commercially available kits (Fujirebio Inc., Shinjuku-ku, Tokyo, 161). T3 suppression test was performed in all of the patients with euthyroid Graves' disease and in some patients with Graves' disease who were euthyroid during antithyroid drug treatment. thyroidal uptake30min after the injection (normal range: %) was assessed before and after a7-days treatment with T3 (75ƒÊg/day). T3suppressibility was considered positive, if the uptake after T3was less than half the pretreatment value and less than1.0%. TBII assay TBII were measured using kits provided by Baxter Travenol Ltd. (Chiyoda-ku, Tokyo, 102), originally prepared by R. S. R. Ltd. (Cardiff CF4 4XN, U. K.) (Southgate et al., 1984). Aliquots of serum (50ƒÊl) were iucubated in duplicate with detergent solubilized TSH receptors (50ƒÊl) for various time periods at25 Ž. 125I-labeled TSH (5,000cpm: 100ƒÊl) was then added and iucubation continued for60min at37 Ž. Assay buffer containing1mg/ml of bovine serum albumin in50mm NaCl; 10mM Tris-HCl ph 7.5; 3mM NaN3 (800ƒÊl) was then added followed by30% W/V polyethylene glycol (mol wt 4000) in1m NaCl to give a final polyethylene glycol concentration of15%. After mixing to homogeneity the tubes were centrifuged (1000 ~g for30min at4 Ž) and the pellets containing receptor bound labeled TSH counted for125i. The amount of125i-labeled TSH non-specifically trapped in the pellet or binding of labeled TSH to test serum was determined by replacing receptors with1% Lubrol in the reaction mixture. Results were expressed as the percentage of inhibition of125i-labeled TSH binding and were calculated as follows: 1- labeled TSH specifically bound in the presence of test sample/ labeled TSH specifically bound in the presence of sample from normal pooled serum When the effect of the length of the second incubation was studied, the incubation was performed for various time periods at37 Ž after the120min preincubation at25 Ž. Bovine TSH was obtained from Armour Pharmaceutical (Chicago, IL60901) and used for the assay after dissolving in the assay buffer. All data were analyzed for statistical significance by Student's t-test or X2analysis. Results When the assay of TBII was performed

3 Vol.34, No.1 IMPROVED ASSAY FOR TBII 15 at various incubation times snd temperatures, the protocol recommended by the manufacturer was found satisfactory except the length of the first incubation during which solubilized receptors were reacted with serum samples. As shown in the right panel of Fig.1, dose-inhibition curves obtained using a Graves' serum showed a gradual increase in the inhibition of labeled TSH binding when the first incubation time was extended from15to240min. On the other hand, no significant difference in the inhibition was observed in the presence of bovine TSH (Fig.1. left panel). In this experiment, 50% inhibition was attained by adding10ƒêl test serum when120min preincubation was done, whereas20ƒêl serum was required in the case of 15 min preincubation which was adopted by Southgate et al. (1984). Thus, 120min preincubation resulted in about a twofold increase in sensitivity compared with the original assay. When inhibition of125i-tsh binding was measured after preincubation of the receptor with Graves' sera for various time periods, it was gradually increased with the incubation time in the presence of all three The effect of the length of the first incubation of test sample with the receptor on inhibition of labeled TSH binding. Dose-inhibition curves for bovine TSH are shown in the left hand figure, and those for 50ƒÊl aliquots of a standard Graves' serum diluted in normal pool serum are shown in the right hand figure. The effect of the length of the first incubation on inhibition of labeled TSH binding in the presence of three Graves' sera ( œ) and various concentrations of bovine TSH ( ).

4 16 KONISHI et al. Endocrinol. February1987 Japon. Graves' sera tested, reaching almost a plateau at around min (Fig.2). Using the same samples and120min preincubation, we studied the effect of the second incubation time (15, 30or60min) on the assay results. Since the binding of to the receptor was time-dependent, total binding at15min was too low to give reliable results. However, the results were found satisfactory after both30and60min incubation. We used60min second incubation in the present study. When the first incubation was performed for15min or120min, mean total binding of125i-labeled TSH to the receptors in the presence of a negative control serum was 25.2 (SD2.5) and22.1 (SD1.2)%, respectively, whereas mean non specific binding was7.5 (SD0.9) and6.9 (SD0.6) Thus, by using a longer incubation period specific labeled TSH binding to the receptor was somewhat decreased. However, reproducibility of the assay was satisfactory as that in the assay using15 min iucubation (Table1). In order to compare the clinical usefulness of assays using15min and120min princubation, TBII assay was carried out using both procedures on199serum samples from normanl subjects and patients with various thyroid disorders. The results obtained by the two methods are shown in Fig.3. The mean TBII values in53 normal subjects obtained using15min and 120min incubation were-2.16 (SD5.91) and-0.46 (SD5.72) %, respectively. TBII exceeding10% in the former condition and 11% in the latter were taken as positive. In patients with both untreated and treated Graves' disease and also with euthyroid Graves' disease, the assay using 120min preincubation yielded a significantly higher inhibition of labeled TSH binding compared to the one using15min preincubation resulting in higher detectability of positive TBII. The increase in the incidence of positive TBII was especially marked in patients with both antithyroid drug treated Graves' disease and euthyroid Graves' disease, Comparison of intra- and inter-assay variation of TBII values obtained using Intra-assay variation (n=5) 15and120min first incubation

5 Vol.34, No.1 IMPROVED ASSAY FOR TBII 17 Comparison of TBII activities obtained using15min and120min preincubation in patients with various thyroid diseases. The shaded areas represent the normal ranges, as defined from the distribution of53normal controls in both assays.

6 18 KONISHI et al. Endocrinol. February1987 Japon. among whom TBII values were generally lower than those in untreated Graves' patients. In euthyroid Graves' disease, all of the5t3-suppressible patients were negative for TBII, whereas among the remaining14t3-nonsuppressible patients, 8 were positive for TBII using120min incubation. On the other hand, 5out of17patients with primary hypothyroidism were positive for TBII. All of the positive patients were nongoitrous. However, there was no significant difference between the TBII activities of these patients using the two methods. Neither was there any significant difference in TBII using the2methods in patients with simple goiter and adenomatous goiter. In patients with thyroid adenoma and cancer TBII were significantly lower using120min incubation than those obtained using15min incubation. One patient with simple goiter who had the lowest TBII value showed a significantly increased binding of125i-labeled TSH to her serum compared to20normal controls. However, no significant labeled TSH binding to serum was found in other patients with low TBII values. The relation of TBII measured by these two methods and the results of the T3 suppression test was studied in49treated patients with Graves' disease. Among32 T3-nonsuppressible patients the incidence of positive TBII increased from19to47% Comparison of the incidence of TBII measured by using15and120min first incubation in T3-nonsuppressible and suppressible patients with Graves' disease receiving maintenance antithyroid drug therapy by Relation of the clinical outcome and TBII at the time of the cessation of the antithyroid drug treatment measured by using120min incubation increasing the preincubation period, while that in T3-suppressible patients changed from18to24% (Table2). Among these patients, 34discontinued antithyroid drugs and were followed up for1year. The relation of the clinical outcome and TBII at the time of the cessation of the treatment is shown in Table3. No significant association between them was observed. Discussion Delayed addition of labeled ligand is known to improve the sensitivity of radioligand assays (Rodbard et al., 1971). Thus, in the radioreceptor assay for TBII developed by Southgate et al. (1984) solubilized TSH receptors were incubated with test serum for15min at20 Ž before the addition of125i-labeled TSH. The present studies revealed that assay sensitivity to TBII was further increased by extending the first inbubation of test serum with the receptors. From the time course of the inhibition of125i-labeled TSH binding, 120 min incubation was considered most appropriate. The sensitivity to TSH was not increased by prolongation of the incubation. The differential effect of the incubation time appears to reflect the difference in binding kinetics between TSH and antibodies reacting against TSH receptors, and may be related with the slow-onset and long-acting nature of bioactivities of these antibodies

7 Vol.34, No.1 IMPROVED ASSAY FOR TBII 19 compared to TSH (McKenzie and Zakarija, 1977). The longer preincubation might cause the damage to the receptors. Actually, a slight decrease in the specific binding of labeled TSH was observed. However, intra- and inter-assay precision of TBII assay using15and120min preincubation were comparable. Furthermore, the range of values obtained with normal sera was similar in the two methods. The second incubation can be shortened to30min instead of60min which is used in the present study. The assay using120min preincubation resulted in a significant increase in inhibition of125i-labeled TSH binding in patients with Graves' disease. TBII detectable in some patients with primary myxedema are considered to be receptorblocking types of antibodies (Konishi, et al., 1985). Specificity of the assay was maintained as was shown by the fact that none of the patients with simple goiter, adenomatous goiter, thyroid adenoma or cancer were positive for TBII. Negative values were obtained in some patients with these disorders. The presence of TSHbinding activities in serum as reported by several investigators (Biro, 1982; Beall and Kruger, 1983; Kajita et al., 1983; Akamizu et al., 1984), was confirmed in a patient with simple goiter. However, the causes of this apparently increased binding of125ilabeled TSH in other cases are unknown. In contrast to the100% detectability of TBII in untreated Graves' patients reported by Southgate et al. (1984), not all the patients we studied were positive for TBII even by using the improved assay. However the assay markedly improved the detectability of TBII in both Graves' patients receiving maintenance antithyroid drug therapy and patients with euthyroid Graves' disease, making it useful for predicting the efficacy of the treatment and for diagnosis of the latter condition. Although TBII are known to decrease after antithyroid drug treatment (Teng and Yeung, 1980; Bliddal et al., 1981), higher detectability of TBII in T3-nonsuppressible patients has been reported by Kuzuya et a/. (1979) as compared with that in Ta-suppressible patients. By using the improved assay, the increase in the detectability of TBII was more marked in T3-nonsuppressible patients than in T3- suppressible patients. Thus, the disappearance of the TBII detectable by this method may serve as a better index for predicting the T3-suppressibility than that by the original assay. Although the TBII incidence was low in euthyroid Graves' disease even when using the improved assay, all TBII-positive patients were T3-nonsuppressible, suggesting a close association of TSH receptor antibodies with thyroid abnormalities in this condition. The findings are similar to those reported by O'Donnell et al. (1978), but not to those by Teng et al. (1977). There is some evidence that TBII determinations could be of prognostic value in predicting the course of Graves' disease (Davies et al., 1977; O'Donell et al., 1978; Schleusener et al., 1978; Teng et al., 1980). However, the data are conflicting (Gardner and Utiger, 1979; Docter, et al., 1980). In spite of the increased detectability of TBII no significant association of TBII with the recurrence was observed in the present study. We are grateful to Baxter Travenol Ltd., for supplying the assay kit, and Miss K. Aoki for secretarial assistance. The investigations were supported by a grant from the Ministry of Education, Science and Culture, Japan. References Akamizu, T., H. Ishii, T. Mori, T. Ishihara, K. Ikekubo and H. Imura (1984). Abnormal thyrotropin-binding immunoglobulins in two patients with Graves' disease. J. Clin. Endocrinol. Metab. 59,

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