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A Coated Tube Assay for the Detection of Blocking Thyrotropin Receptor Autoantibodies

MiLo GmbH (W.B.M.), Bioassays GmbH (C.L.), and Research Department (A.B., N.G.M.), B.R.A.H.M.S. AG, Biotechnology Center Hennigsdorf/Berlin, D-16761 Hennigsdorf, Germany

Address all correspondence and requests for reprints to: Dr. Nils G. Morgenthaler, Research Department, B.R.A.H.M.S. AG, Biotechnology Center Hennigsdorf/Berlin, Neuendorfstr. 25, D-16761 Hennigsdorf, Germany. E-mail: n.morgenthaler{at}brahms.de.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We developed a coated tube assay to discriminate TSH-receptor-stimulating autoantibodies [thyroid-stimulating antibodies (TSAb)] from those autoantibodies blocking TSH binding without intrinsic activation [thyroid-blocking antibodies (TBAb)]. The wild-type TSH receptor in the TSH binding-inhibitory assay was exchanged for a chimeric receptor where a TSAb epitope (amino acids 8–165) was replaced by comparable LH-R residues. Binding of ¹²⁵I-labeled TSH to this chimera could be inhibited by sera containing TBAb up to 95%. Sera from 316 patients with Graves’ disease and 17 with autoimmune thyroid disease were grouped according to their bioassay activity. At the decision threshold, the chimera A assay had a sensitivity of 78.0% for TBAb with a specificity of 90.2%. In detail, 19 of 22 (86.4%) TBAb sera and 15 of 23 (65.2%) TSAb/TBAb sera were positive but only 32 of 216 (14.0%) TSAb sera and 5 of 72 (6.9%) bioassay negative sera. There was a weak but significant positive correlation (r = 0.46) between the chimera assay and the bioassay for TBAb. This is the first report of a coated tube assay for the determination of TBAb employing an adaptation of the TSH binding-inhibitory format, which could be a useful alternative to the bioassay.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
THE TSH RECEPTOR (TSH-R) is the target autoantigen in autoimmune thyroid diseases. TSH-R autoantibodies (TRAb) are functionally heterogeneous; they include both thyroid-stimulating antibodies (TSAb) as well as thyroid-blocking antibodies (TBAb). Stimulating autoantibodies activate the receptor and cause hyperthyroidism. More rarely, receptor occupancy by nonstimulatory blocking TSH-R autoantibodies prevents TSH action and may cause hypothyroidism. Both TSAb and TBAb together comprise the TSH binding-inhibition (TBII) activity (see Ref. 1 for review). The precise epitopes for TSAb or TBAb are still under debate, but there were reports that an important functional epitope for TSAb exists in the N-terminal region of the extracellular domain of TSH-R, between amino acid residues 25 and 165, and a major TBAb epitope was described in the C terminus of the extracellular domain of TSH-R, between amino acid residues 261–370, respectively (2, 3, 4). However, other studies suggest that TSAb, TBAb, and TSH share epitopes in close vicinity (5).

The detection of TRAb is possible with a variety of assay systems (reviewed in Refs. 6 and 7), but the only assay widely used in clinical practice is the competitive TBII assay, based on the interaction between bovine TSH and autoantibodies (8). Although this assay system could be improved to detect TRAb in 98% of patients with Graves’ disease (GD) (9), it does not discriminate between different activities of autoantibodies. So far, TSAb and TBAb can only be detected in bioassays of TSH-R-mediated cAMP synthesis. Unfortunately, the use of bioassays is limited because of the need of cell culture facilities. A perspective strategy for the detection of different activities of TSH-R autoantibodies is the use of TSH-R chimeras, wherein epitopes for TSAb or TBAb are replaced by lutropin/choriogonadotropin (LH-CG) receptor sequences (4, 10, 11).

We report, in this study, on a coated tube assay for the discrimination of TBII-positive sera in those with TBAb activity, using a TSH/LH-CG receptor chimera, wherein a well-described TSAb epitope is replaced by a comparable LH-CG receptor residue. This chimera retains high affinity to TSH and thus can be used for the detection of TBAb in a TBII assay format.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Patient sera

GD sera (n = 316) were obtained from blood donors recruited for the development of in vitro diagnostics (Invent GmbH, Biotechnology Center Hennigsdorf/Berlin, Germany). This blood donation was approved by a national ethical committee. GD was defined, on clinical terms, by a physician and confirmed by antibody detection in the human recombinant TBII assay (DYNOtest TRAK human, B.R.A.H.M.S. AG). All patients were under treatment at the time of blood withdrawal (median, 5.4 months; range, 1–12 months). Sera (n = 17) from patients with autoimmune thyroid disease, who were clinically hypothyroid but contained high levels of TBII, are described elsewhere (12) (gift from Dr. Daphne Khoo, Singapore). Control sera (n = 100) were obtained from individuals with no personal or family history of endocrine autoimmune disease. Sera were negative for autoantibodies to TSH-R, thyroid peroxidase, and thyroglobulin. Written consent was given by all blood donors.

Standard assays for TRAb detection

TBII assay with wild-type (WT) human recombinant TSH-R (affinity immobilized on antibody coated tubes) was performed as described (9). The technical and clinical evaluation of this assay (DYNOtest TRAK human, B.R.A.H.M.S. AG, Hennigsdorf, Germany) is described in detail elsewhere (13). The assay is calibrated 1:1 to the World Health Organization standard 90/672. Data are expressed in percentage inhibition of TSH binding or, alternatively, in international units (IU/liter). TSAb detection was carried out using unfractionated sera in Chinese hamster ovary cells transfected with the human TSH-R and a cAMP-responsive luciferase gene as described previously (14). Luciferase activity was determined by a luminometer (Berthold, Germany). Thyroid stimulation index (SI) was calculated as: SI (%) = 100 x [relative light units (RLU) patient/RLU euthyroid control]. SI more than 1.5 was considered positive. For TBAb detection, bovine TSH (1 mU/ml, Sigma) was added, either with euthyroid control or with test serum, as described (15); and the luciferase activity of a test serum and a pool of euthyroid control serum, both in the presence of TSH, was compared. The inhibition index (InI) was calculated as: InI (%) = 100 x [1–(RLU patient/RLU euthyroid control)]. An InI more than 40% was considered positive.

Chimeric TSH-R-coated tube assay for TBAb detection

Construction of TSH/LH-CG receptor chimera (chimera A). TSH/LH-CG receptor chimera was constructed similar to the approach of Tahara et al. (4, 10) and is described in detail elsewhere (11). Briefly, the N-terminal part of the human TSH-R cDNA (16) was substituted with amino acids 1–165 of the rat LH-CG receptor (gift of Dr. Segaloff, University of Iowa, Iowa City, IA). The resulting chimeric receptor was termed chimera A (11).

Generation of chimera-A-producing cells. HEK 293 cells were grown in DMEM (Life Technologies, Inc., Karlsruhe, Germany) supplemented with 10% fetal bovine serum. Cells were cultivated in a 5% CO2 atmosphere at 37 C. HEK 293 cells were transfected with pIRESneo-vector containing chimera A using SuperFect (Qiagen, Hilden, Germany) transfection reagent according to the manufacturer’s instruction. Forty-eight hours after transfection, selection was started with 0.8 mg/ml G418 (Life Technologies, Inc.). After cloning by limiting dilution, a stable clone expressing high levels of chimera A (about 10⁶ receptors per cell) was selected. The amount of TSH-R was determined by comparison of TSH binding activity of HEK293 cell extracts and extracts prepared from the characterized K562 TSH-R cells (9) (data not shown).

Preparation of chimera A cell extract. Confluent 293 cells grown in a 75-cm² plate were resuspended by scraping into PBS and were washed twice in the same buffer by centrifugation at 2,500 rpm. The resulting cells were lysed in 0.5 ml buffer containing 20 mM HEPES-KOH (pH 7.5), 50 mM NaCl, 1% Triton X-100, 10% glycerol. The suspension was centrifuged at 30,000 x g for 1 h; the supernatant (8 mg/ml total protein) was collected and stored at -70 C.

Production of chimera-A-coated tubes. Donkey antisheep polyclonal antibodies (Sigma, St. Louis, MO) were coated for 20 h on polystyrene tubes (7 µg/ml in 0.3 ml buffer with 20 mM NaCl, 20 mM HEPES-KOH, pH 7.5). Tubes were washed with the same buffer and incubated for 20 h in 0.3 ml of the same buffer containing 1.7 µg/ml sheep polyclonal antibodies (B.R.A.H.M.S. AG) to amino acids 737–758 of the TSH-R. The chimera A containing cell extract was diluted 1:50 with buffer (100 mM HEPES-KOH, 0.5% Triton X-100, 0.5% BSA, pH 7.5) and added to the tubes. Affinity binding of the chimera A to the antibody was performed at 4 C for 20 h. Tubes were blocked (50 mM HEPES-KOH, 0.1% Triton X-100, 0.5% BSA, pH 6.5) and lyophilized.

Autoantibody measurement in chimera A TBII assay. Patients’ sera or standards (100 µl) were added in duplicate to chimera-A-coated tubes. To this was added 200 µl buffer containing 100 mM HEPES-KOH, 1% BSA, 0.5% Triton X-100, 5 µg antihuman TSH antibody, protease inhibitor cocktail (Boehringer, Mannheim, Germany), pH 7.5. After 2 h incubation, under shaking at room temperature, tubes were washed twice with 2 ml washing buffer. Then, 200 µl (1 ng) bovine ¹²⁵I-labeled TSH was added (56 Ci/g, B.R.A.H.M.S. AG), followed by 1 h incubation at room temperature. Tubes were washed three times with 2 ml washing buffer, and radioactivity was counted. The TBII activity of the sera was presented as percentage inhibition calculated as: InI % = 100 x [1 - (cpm test serum/cpm control serum pool)], where cpm represents counts per minute. To obtain the optimal decision threshold level for positivity in the chimera A assay, receiver-operating characteristic (ROC) analysis was performed (17). Sensitivity (the true-positive results) was calculated from 45 patients with confirmed TBAb in the bioassay (>40% InI), whereas specificity (the true-negative results) was calculated from all patients and controls with absent TBAb in the bioassay.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Chimera A was produced in stable transfected HEK 293 cells. Addition of 1 mU/ml bovine TSH to 10⁴ HEK 293 cells/well increased cAMP production to 1081 ± 111 fmol/well, compared with 26 ± 3.8 fmol/well with buffer alone (42-fold stimulation). In Chinese hamster ovary cells with WT TSH-R, the stimulation was 37-fold. When ¹²⁵I-labeled bovine TSH was added to both types of cells, specific binding could be displaced by increasing concentrations of unlabeled TSH in a comparable manner (data not shown). Thus, the affinity for bovine TSH and the ability to produce cAMP as a result of this stimulus were comparable between WT TSH-R and Chimera A, as already shown by others (4, 10).

After detergent extraction, chimera A was immobilized on polystyrene tubes. The specific binding of ¹²⁵I-labeled bovine TSH to chimera A was preserved and indistinguishable from WT TSH-R. When tubes were preincubated with normal euthyroid sera, about 10–11% of the total TSH tracer added (100,000 cpm) bound to chimera A or WT TSH-R. The nonspecific binding in the absence of chimera A or WT TSH-R was 0.1–0.2% (Fig. 1). The specific binding could be inhibited by excess cold TSH to background levels (99% inhibition) for both receptors. However, when we tested three sera from patients with GD (s1, s2, and s3) and from patients with autoimmune hypothyroidism (b1, b2, and b3) in both assays, inhibition of TSH binding to WT TSH-R was seen with all six sera, but inhibition of tracer binding to chimera A was only seen in the hypothyroid sera (Fig. 1).

View larger version (17K): [in this window] [in a new window]   FIG. 1. Binding of TSH and TSH-R antibodies to WT TSH-R and chimera A. Wild-type (WT) TSH-R (black bars) and chimera A (gray bars) were coated on polystyrene tubes (see Subjects and Methods) and incubated with 100,000 cpm 125I-labeled bovine TSH. Groups are labeled: no receptor, background cpm in the absence of TSH-R or chimera A; control serum, specific binding of TSH in the presence of a pooled serum from healthy blood donors; control serum + TSH, maximal suppression of binding by excess unlabelled TSH; b1–b3, binding of TSH in the presence of sera containing TBAb; s1–s3, binding of TSH in the presence of sera containing TSAb.

View larger version (20K): [in this window] [in a new window]   FIG. 2. Inhibition of TSH binding to chimera A by sera containing TBAb or TSAb. All sera were tested in the chimera A assay (A) and WT TSH-R (B) for their effect on TSH binding. Sera were grouped according to their bioactivity in the TSH-R bioassay in those having no detectable bioactivity (bioassay neg.), those with TSAb activity, those with TSAb and TBAb activity, and those with only TBAb activity. Data are expressed as percentage inhibition of TSH binding. Median is indicated by a solid line. The horizontal dotted line indicates the cut-off levels.

A closer look at the individual groups revealed an increase in the percentage of chimera A-positive sera from the bioassay negative group to the TBAb group. This increase was highly significant (P < 0.001, ² test), between the negative or TSAb group and the TSAb/TBAb or TBAb group. In detail, 5 of 72 (6.9%) bioassay negative sera were positive in the chimera A assay, 32 of 216 (14.0%) TSAb sera, 15 of 23 (65.2%) TSAb/TBAb sera, and 19 of 22 (86.4%) TBAb sera. In the WT TSH-R assay, all tested sera were positive, which was an inclusion criteria of the study, because a WT negative serum was unlikely to bind to chimera A. There was no significant difference in free T₃, T₄, or TSH levels in those patients with TSAb who were still positive in the chimera A assay, compared with those patients not reacting with chimera A.

Comparing both WT TSH-R assay and chimera A assay for each group, the WT TSH-R assay resulted in significantly higher inhibition values than the chimera A assay (P < 0.0001 by Wilcoxon) in all groups. As shown in Fig. 3 for individual TSAb sera, the often very high inhibition in the WT TSH-R assay was lost in the chimera A assay for the majority of sera. The overall correlation between the WT and the chimera A assays was r = 0.47 (n = 333; 95% CI, 0.38–0.55; P < 0.0001; Fig. 3), and the correlation between the chimera A assay and the bioassay for TBAb was r = 0.46 (95% CI, 0.32–0.58; P < 0.001). There was no significant correlation between chimera A and TSAb bioassay data (r = 0.02). Also, most sera negative in the chimera A-inhibition assay were also negative for cAMP production on chimera A-expressing HEK 293 cells (data not shown).

View larger version (39K): [in this window] [in a new window]   FIG. 3. Correlation of WT TSH-R and chimera A data. Shown is the Spearman rank correlation for all 333 sera (r = 0.47; 95% CI, 0.38–0.55; P < 0.0001). The inset shows individual TSAb sera from Fig. 2 in both assays. The often very high reactivity with the WT TSH-R was lost on chimera A for the majority of sera.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

In the present study, we used this chimera to analyze sera from patients with GD and other autoimmune thyroid diseases in a coated tube assay. The coated tube technology allows quick, simple, and reproducible analysis of test sera and is technically superior to bioassays. Overall, our data allow a differentiation between sera containing TBAb or TBAb together with TSAb on one side, and sera with only TSAb or no bioactivity on the other. This was seen for the median level of inhibition and the number of chimera A positive sera in each group. We also found a weak but significant positive correlation between results obtained in the chimera A assay and the bioassay for TBAb. Most TBAb sera (86.4%) and 65% of those sera containing TBAb together with TSAb were positive in the chimera A assay. The high negative predictive value of 97.3% suggests a clinical usefulness, at least for those patients whose serum is negative for chimera A.

However, it cannot be ignored that 14% of TSAb sera and 7% of sera without any bioactivity were also positive at the ROC plot obtained cut-off. There are two possible explanations for this observation. The first, and probably favored by the current concept, is the presence of TBAb in those TSAb-positive sera. However, we tried to account for this, by putting all sera with a mixture of TSAb/TBAb in a different group. In this group, the percentage of sera positive with chimera A was significantly higher than in the TSAb group. Furthermore, the definition of this mixture of antibodies is far from clear. Dealing with these antibodies, one must consider that only TSAb properties are intrinsic (hence, the result of an interaction between antibody and TSH-R), whereas TBAb is detected indirectly by reducing a TSH effect in the assay system. Instead of containing bona fide TBAb, these so-called mixed sera might as well contain antibodies that show weak TSAb but strong TSH competing properties. By definition of a bioassay, this sera would be classified as TBAb positive, although no intrinsic effect on the TSH-R is apparent.

We favor the explanation that the chimera-A-positive sera from the TSAb group interact with those parts of the extracellular domain of TSH-R, which is still conserved on chimera A. These data are in agreement with observations showing the epitope heterogeneity of TSH-R autoantibodies (18, 19).

Nevertheless, there is also the concept suggesting that TSAb, TBAb, and TSH share epitopes in close vicinity (5), which was recently supported in a few sera by displacement studies with monoclonal antibodies (20, 21, 22). Maybe reactivity with chimera A is not merely a matter of epitope recognition but due to very high TBII levels in TBAb-positive patients. Although this cannot be excluded, we see no reactivity to chimera A in many TBAb-negative patients with equally high TBII levels (Fig. 3). Also, reactivity to chimera A was still present in TBAb patients after dilution (data not shown). The problem of epitope specificity of different classes of TRAb is still an open question and needs further investigation in more detail, maybe with purified TRAb from human serum and only slightly modified chimeras (23).

We propose that the chimera-A-coated tube assay, despite its limitations, may contribute to the detection and investigation of TBAb in a bioassay-independent system. This might be useful for the subtype analysis of TSAb and TBAb in patients’ sera and could help to elucidate the enigma of TRAb-TSH-R interaction.

	Acknowledgments

 
The authors thank Dr. J. Struck and Mr. T. Chen (B.R.A.H.M.S. AG, Hennigsdorf, Germany) for kindly providing antibodies to C terminus of TSH-R and for helpful discussions. We express appreciation to Dr. U. Loos (University of Ulm, Ulm, Germany) for the concept of using a chimera similar to the Mc1 + 2 chimera. We thank Dr. D. L. Segaloff (University of Iowa, Iowa City, IA) for the pcDNA3-rLHR(B9) plasmid.

	Footnotes

 
This work was supported by a grant from the European Union and the State of Brandenburg (Verfahrensinnovation no. 80084492) to MiLo GmbH.

Abbreviations: CI, Confidence interval; GD, Graves’ disease; LH-CG, lutropin/choriogonadotropin; ROC, receiver-operating characteristic; TBAb, thyroid-blocking antibodies; RLU, relative light units; TBII, TSH binding inhibition; TRAb, TSH-R autoantibodies; TSAb, thyroid-stimulating antibodies; TSH-R, TSH receptor; WT, wild-type.

Received May 12, 2003.

Accepted October 2, 2003.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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