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Polyethylene glycol increases the detection of anti-thyrotropin receptor antibodies by a radioreceptor assay.

Autoantibodies to the thyrotropin (TSH) receptor (TSHR) are the hallmark of the autoimmune response to the thyroid gland and are responsible for thyrotoxicosis in Graves disease (1). Unlike other autoantibodies, there is no direct assay for TSHR antibodies. Instead, these antibodies are detected either by their ability to inhibit binding of radiolabeled TSH to receptors or by a bioassay of TSHR activation (2, 3). The most widely used assay is a radioreceptor assay, which uses solubilized porcine TSHR, in which the antibody is called TSH-binding inhibitory immunoglobulin (TBII). Serum TBII is easily assayed with a commercially available kit; however, 5-10% of patients with Graves disease show a negative reaction perhaps related to inadequacies of the assay (4).

Addition of polyethylene glycol (PEG) enhances cAMP production induced by a patient's IgG, but not by TSH, forskolin, or GTP[gamma]S in an in vitro bioassay for thyroid stimulators (5), suggesting that the enhancement occurs at the step of antibody-receptor interaction. We recently found that the modification of TSHR with detergent changed the nature of the binding of TSHR to anti-TSHR antibodies (6). On the basis of these findings, we examined whether the addition of PEG increase the ability of the TBII assay to detect autoantibodies to the TSHR.

The TBII assay was performed as described previously (4, 6). Serum samples (50 [micro]L) were incubated in duplicate with solubilized porcine TSHR (50 [micro]L) for 15 min at room temperature (first incubation). [sup.125]I-labeled bovine TSH (50 [micro]L) and assay buffer (100 [micro]L) were then added, and the incubation was continued for 60 min at 37[degrees]C (second incubation). Six hundred microliters of 300 g/L PEG (average Mr 6000), 250 [micro]L of assay buffer, and 50 [micro]L of 500 g/L PEG were then added to the incubated mixture. The mixture was then centrifuged at 2000g for 20 min at 4[degrees]C, and the radioactivity in the pellets was measured. TBII activity was expressed as inhibition of labeled TSH binding (4).

To examine the effect of PEG on TBII activity, 50 [micro]L of 500 g/L PEG, 50 [micro]L of [sup.125]I-TSH, and 50 [micro]L of assay buffer were added to the mixture before the second incubation (final concentration of PEG was 100 g/L). After the mixture was incubated for 60 min at 37[degrees]C, 600 [micro]L of 300 g/L PEG and 300 [micro]L of assay buffer were added, the radioactive fraction was precipitated, and the radioactivity was measured.

[FIGURE 1 OMITTED]

In samples from 24 untreated patients with Graves disease and positive TBII (group A in Fig. 1), the TBII activity increased significantly (P <0.0001) after the addition of PEG. We next examined 17 untreated patients with Graves disease and negative TBII (by conventional assay). These patients were thyrotoxic, and the diagnosis was confirmed by normal or increased radioactive iodine uptake (Table 1). When samples from these patients were assayed in the sensitive TBII assay with 100 g/L PEG, TBII became positive in 14 (82%) of these patients (group B in Fig. 1). On the other hand, none of the 16 healthy subjects showed a positive reaction even with this sensitive TBII assay (group C in Fig. 1). All patients with other autoimmune diseases (n 5 11), including rheumatoid arthritis (n 5 3), were also negative in the sensitive assay.

PEG, a nonionic hydrophilic polymer, has long been known to enhance the reaction rate and sensitivity of immunoassays (7). PEG is also used as a promoter of cell fusion and is known to reorient membrane proteins and lipids (8). In the TBII assay, PEG may have modified the three-dimensional configuration of TSHR and enhanced the binding activity of anti-TSHR antibodies.

The present study showed that the sensitive TBII assay with PEG enabled the diagnosis of patients with Graves disease whose TBII could not be detected by conventional assays. Furthermore, this sensitive TBII assay may be useful for diagnosing disease remission, especially during antithyroid drug therapy. In our previous study, one-half of the patients relapsed with thyrotoxicosis after the therapy was discontinued when TBII became negative by the conventional assay (9). Similar results were also reported by other groups (2). At the present time, no method is available to predict which patients will relapse. Additional study is needed to determine whether the sensitive TBII assay with PEG can differentiate between patients who will relapse and those who will not.

This work was supported by a grant-in-aid for Scientific Research (No. 09307055 to N.A.) from the Ministry of Education, Science and Culture of Japan.

References

(1.) Prabhakar BS, Fan JL, Seetharamaiah GS. Thyrotropin-receptor mediated disease: a paradigm for receptor autoimmunity. Immunol Today 1997;18: 437-42.

(2.) Feldt-Rasmussen U, Schleusener H, Carayon P. Meta-analysis evaluation of the impact of thyrotropin receptor antibodies on long term remission after medical therapy of Graves' disease. J Clin Endocrinol Metab 1994;78:98-102.

(3.) Watanabe Y, Tahara K, Hirai A, Tada H, Kohn LD, Amino N. Subtypes of anti-TSH receptor antibodies classified by various assays using CHO cells expressing wild type or chimeric human TSH receptor. Thyroid 1997;7:13-9.

(4.) Tamaki H, Amino N, Watanabe Y, Aozasa M, Hayashi H, Tachi J, et al. Radioreceptor assay of anti-TSH receptor antibody activity: Comparison of assays using unextracted serum and immunoglobulin fractions, and standardization of expression of activities. J Clin Lab Immunol 1986;20:1-6.

(5.) Inui T, Kouki T, Yamashiro K, Hachiya T, Ochi Y, Kajita Y, et al. Increase of thyroid stimulating activity in Graves' immunoglobulin-G by high polyethylene glycol concentrations using porcine thyroid cell assay. Thyroid 1998;8: 319-25.

(6.) Watanabe Y, Tada H, Hidaka Y, Takano T, Amino N. Effect of solubilization of porcine thyrotropin (TSH) receptor on TSH binding and on radio-receptor assay for anti-TSH receptor antibodies. Biochem Biophys Res Commun 1998;248:110-4.

(7.) Lizana J, Hellsing K. Polymer enhancement of automated immunological nephelometric analysis, as illustrated by determination of urinary albumin. Clin Chem 1974;20:415-20.

(8.) Strasser RH, Lefkowitz RJ. Homologous desensitization of [beta]-adrenergic receptor coupled adenylate cyclase. Resensitization by polyethylene glycol treatment. J Biol Chem 1985;260:4561-4.

(9.) Tachi J, Amino N, Tamaki H, Takeoka K, Kimura M, Iwatani Y, et al. Cellular immunity as a valuable factor for prognostic prediction in patients with Graves' disease under antithyroid drug therapy. Thyroidology 1989;2:85-92.

Assay, Yukihiko Watanabe, [1] Hisato Tada, [1] Yoh Hidaka, [2] Toru Takano, [1] Keiko Takeoka, [1] Shuji Fukata, [2] Kanji Kuma, [2] and Nobuyuki Amino [1] * ([1] Department of Laboratory Medicine, Osaka University Medical School, Suita 565-0871, Japan, and [2] Kuma Hospital, Kobe 650-0011, Japan; * author for correspondence: fax 81-6-879-3239, e-mail namino@labo. qjmed.osaka-u.ac.jp)
Table 1. Pertinent laboratory data for untreated patients with
Graves disease and negative TBII (group B).

Patient Age, Sex [FT.sub.4],
No. years (a) pmol/L

 1 49 F 57.8
 2 28 M 45.9
 3 68 F 36.9
 4 46 M 46.4
 5 14 F 33.3
 6 33 M 41.8
 7 18 F 64.4
 8 45 M 36.5
 9 25 F 44.3
10 31 F 49.4
11 31 F 29.0
12 58 F 43.2
13 56 M 59.0
14 35 F 43.1
15 61 F 48.3
16 48 F 70.2
17 22 F 49.9
Reference values 9.7 - 23.2

Patient [FT.sub.3], TSH, RAID, %
No. pmol/L milliunits/L

 1 18.5 <0.07 46.8
 2 13.3 <0.07 42.4
 3 10.6 <0.07 40.6
 4 13.7 <0.07 36.4
 5 9.1 <0.07 31.0
 6 17.0 <0.07 37.7
 7 20.0 <0.07 31.1
 8 15.7 <0.07 36.6
 9 14.3 <0.07 47.4
10 16.3 <0.07 56.2
11 9.9 <0.07 45.0
12 18.5 <0.07 30.3
13 18.5 <0.07 41.8
14 14.5 <0.07 22.4
15 19.9 <0.07 42.0
16 21.7 <0.07 37.4
17 15.2 <0.07 17.2
Reference values 3.6 - 7.0 4.0 - 4.5 10 - 40

Patient TGHA MCHA
No.

 1 Negative Negative
 2 Negative Negative
 3 Negative Negative
 4 Negative Negative
 5 Negative 1600
 6 Negative Negative
 7 100 6400
 8 Negative 400
 9 400 100
10 100 1600
11 Negative 1600
12 100 400
13 1600 6400
14 Negative 25600
15 Negative 1600
16 Negative 1600
17 Negative 400
Reference values Negative Negative

(a) [FT.sub.4], free thyroxine; [FT.sub.3], free triiodothyronine;
RAID, radioactive iodine uptake; TGHA, anti-thyroglobulin
hemagglutination antibody; MCHA, anti-thyroid microsomal antibody.
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Title Annotation:Technical Briefs
Author:Watanabe, Yukihiko; Tada, Hisato; Hidaka, Yoh; Takano, Toru; Takeoka, Keiko; Fukata, Shuji; Kuma, Ka
Publication:Clinical Chemistry
Date:Mar 1, 1999
Words:1466
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