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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 7 2129-2134
Copyright © 1997 by The Endocrine Society

Experimental Studies

The Human Thyrotropin (TSH) Receptor in a TSH Binding Inhibition Assay for TSH Receptor Autoantibodies1

Ayumu Kakinuma, Gregorio D. Chazenbalk, Juan Carlos Jaume, Basil Rapoport and Sandra M. McLachlan

Thyroid Molecular Biology Unit, Veterans Administration Medical Center and the University of California, San Francisco, California 94121

Address all correspondence and requests for reprints to: Sandra M. McLachlan, Ph.D., Veterans Administration Medical Center, Thyroid Molecular Biology Unit (111T), 4150 Clement Street, San Francisco, California 94121.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Seven years after the molecular cloning of the human TSH receptor (TSHR), the porcine TSHR remains in general use in the TSH binding inhibition (TBI) assay for autoantibodies to the TSHR. We compared porcine and recombinant human TSHR in two types of TBI assays: one using intact Chinese hamster ovary cells expressing the recombinant human TSHR on their surface, and the other using soluble receptors extracted from these cells with detergent. In the intact cell TBI assay, monolayers expressing large numbers of TSHR were less effective than cells expressing few receptors. These findings are consistent with the very low concentration of TSHR autoantibodies in serum. Binding of [125I]human TSH was about 5-fold lower than that of [125I]bovine TSH to the intact cells. Nevertheless, TBI values with the two ligands were similar for most sera. However, a few sera produced greater inhibition of human than of bovine TSH binding. In the solubilized human TSHR TBI assay, in contrast to the intact cell TBI assay, cells expressing very large number of TSHR were an excellent source for detergent extraction of soluble human TSHR, but only if the cells were extracted while still on the dish and not after scraping. A 10-cm diameter dish of cells provided TSHR for 100–200 replicate determinations when substituted for solubilized porcine TSHR in a commercial TBI kit. TBI values in serum from 30 individuals with suspected Graves’ disease correlated closely when tested with solubilized human and porcine TSHR (r = 0.954; P < 0.001). However, 2 sera that were negative with the porcine TSHR were positive with the human TSHR. TBI and thyroid-stimulating activity in these sera correlated weakly regardless of whether the TBI used human or porcine TSHR. These findings open the way to a practical TBI assay using recombinant human TSHR.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AUTOANTIBODIES to the TSH receptor (TSHR) are the hallmark of the autoimmune response to the thyroid gland in Graves’ disease (reviewed in 1 . In most cases, these TSHR autoantibodies activate the receptor and lead to hyperthyroidism. More rarely, receptor occupancy by nonstimulatory TSHR autoantibodies can prevent TSH action and cause hypothyroidism (2, 3, 4). Unlike autoantibodies to other thyroid autoantigens (thyroid peroxidase and thyroglobulin), there is at present no direct clinical assay for TSHR autoantibodies. Instead, these autoantibodies are detected either by their ability to inhibit radiolabeled TSH binding [TSH binding inhibition (TBI)] or in a bioassay of TSHR activation [TSHR stimulatory immunoglobulin assay (TSI)] (reviewed in 5 . The most widely used assay is a TBI involving porcine TSHR solubilized with detergent from thyroid glands (6).

The molecular cloning of the TSHR complementary DNA (cDNA) led to the expectation that TBI assays using recombinant human (h) TSHR would soon arise. However, although mammalian cell lines stably transfected with the hTSHR cDNA have been established (7, 8, 9, 10, 11, 12), and large amounts of receptor protein have been generated in bacteria (13, 14, 15, 16, 17, 18) and insect cells (19, 20, 21) or as cell-free translates (22), recombinant hTSHR has not yet supplanted the use of porcine TSHR in the TBI assay.

TSHR expressed in mammalian cells are well recognized by autoantibodies in TBI assays involving intact cells (9, 23), cell particulate fractions (10, 24, 25), and detergent-solubilized membranes (25). However, assays using cultured cells are impractical for general use. Further, the small amount of recombinant TSHR recovered from mammalian cell particulate fractions (10, 25) makes use of this material prohibitively expensive. In the present study, we have evaluated the TBI assay using recombinant hTSHR from three stably transfected Chinese hamster ovary (CHO) cell lines.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures

We used three different, stably transfected, CHO cell lines expressing approximately 16,000 (7, 26), 150,000 (26), and 1.9 x 106 (27) TSHR on the cell surface (summarized in Table 1Go). All cell lines (cloned by limiting dilution in selection medium) were grown in Ham’s F-12 medium supplemented with 10% FCS and standard antibiotics. Cells were cultured to confluence in either 10-cm diameter, 24-well cluster or 96-well microtiter culture dishes, as described in the text.


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  		Table 1. Characteristics of cell lines expressing the human TSH receptor

 
Sera

Forty-two sera were used. All were provided by Mr. Juan Tercero of Corning Nichols Institute (San Juan Capistrano, CA), a reference laboratory to which the sera were sent for known or suspected Graves’ disease. Sera were selected to provide a balanced spectrum of TBI values (high, medium, and low or negative) as determined by Corning Nichols. Sera were reassayed in our laboratory as described below.

TBI assay using intact cells

Highly purified bovine (b) TSH (NIH) or recombinant hTSH (Sigma Chemical Co., St. Louis, MO; 5 µg) was radiolabeled with 125I to a specific activity of about 80 µCi/µg protein using the Bolton-Hunter reagent (DuPont-New England Nuclear, Boston, MA), as described previously (26). Four-kilobase (4kb) TSHR and TSHR-0 cells were grown to confluence in 24- and 96-well culture dishes, respectively. TBI activity was determined in a two-step assay, as described previously, using polyethylene glycol (PEG)-precipitated IgG (23) with the following modifications. Cells were preincubated for 1.5 h at 37 C with 0.25 mL (24-well plate) or 0.1 mL (96-well plate) of the IgG preparation in phosphate-buffered saline. In some assays using 96-well plates, cells were preincubated in whole serum (50 µL) before rinsing and the addition of [125I]TSH. With the two-step assay, we found no difference between whole serum and PEG-precipitated IgG (data not shown). After two rinses with binding buffer (Hanks’ buffer containing 280 mmol/L sucrose instead of NaCl and supplemented with 0.25% BSA) (28), [125I]TSH in binding buffer was added to the cells (2 h at 37 C) in the amounts described in the text (250 and 50 µL/well in the 24- and 96-well plates, respectively). Bound [125I]TSH was measured as previously described (23). Nonspecific binding to untransfected CHO cells was subtracted to obtain values for specific binding.

Solubilized TSHR preparation

Receptors were prepared from TSHR-10,000 cells in two procedures.

Cells removed from the culture dishes. Fifty confluent 10-cm diameter dishes of cells (107 cells/dish) were rinsed once with phosphate-buffered saline, and cells were resuspended by scraping into buffer A \[10 mmol/L Tris (pH 7.5), 0.1 mg/mL phenylmethylsulfonylfluoride, 1 µg/mL leupeptin, 1 µg/mL aprotinin, and 2 µg/mL pepstatin A; Sigma; 3 mL/dish). After brief homogenization with a Polytron (10 s, three times, at 4 C), the 500–20,000 x g particulate fraction was processed according to the protocol of Rees Smith et al. (6, 29). The final extraction was performed with 5 mL of 10 mmol/L Tris (pH 7.5), 50 mmol/L NaCl, and 1% Triton X-100. This material was used for TSH binding either directly or after dilution in the same buffer, as described in the text.

Direct extraction of cells in monolayer. Culture medium in one confluent 10-cm diameter dish of cells was removed and replaced with 3 mL of the 1% Triton X-100 buffer described above, supplemented with 5 mmol/L ethylenediamine tetraacetate, 5 mmol/L ethyleneglycol-bis-(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 5 mmol/L N-ethylmaleimide, 10% glycerol, 0.5% BSA, and 0.5% gelatin. After rocking for 2 h at 4 C, the buffer was recovered and centrifuged (1 h, 100,000 x g), and the supernatant was used in the TBI assay as described above. The supplements to the buffer did not alter TSH binding in the assay. In later experiments, we observed that 10 mmol/L Tris (pH 7.5), 50 mmol/L NaCl, 1% Triton X-100, and 0.55 BSA, without the other ingredients, were sufficient for effective TSHR extraction.

TBI assay using solubilized TSHR

Sera were assayed using TSHR antibody kits purchased from Kronus (San Clemente, CA). Reagents from this kit (RSR, Cardiff, UK) were also used in conjunction with solubilized hTSHR, obtained as described above. TBI values were calculated as follows:

TSI assay

TSHR-0 cells, grown to confluence in 96-well culture plates, were assayed as previously described for human thyroid cells (30, 31). This modified procedure uses hypotonic medium (32). For this study, the following additional modifications were introduced. IgG was precipitated with PEG (see above) and resuspended in the hypotonic medium supplemented with 10 mmol/L HEPES (pH 7.4), 1 mmol/L 3-isobutyl-1-methylxanthine, and 0.3% BSA. Cells were incubated in this medium (0.1 mL) for 2 h at 37 C. cAMP in the medium, diluted in 50 mmol/L Na acetate, pH 6.2, and acetylated (31), was measured by RIA using cAMP, 2'-O-succinyl-[125I\]iodotyrosine methyl ester (DuPont, Boston, MA), and a rabbit anti-cAMP antibody (Calbiochem, San Diego, CA). TSI activity was expressed as a percentage of the cAMP value in the test serum relative to cAMP measured after concurrent incubation with sera from normal individuals.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The TBI assays performed were of two main types (Table 2Go) involving either intact CHO cells or solubilized TSHR.


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  		Table 2. Types of TSH binding inhibition (TBI) assays performed

 
TBI assay using cells in monolayer

Previously, we reported data from a TBI assay using intact CHO cells stably transfected with the full-length 4kb hTSHR cDNA (23). The cells used in this assay (7), which expressed about 16,000 receptors/cell (26), were equal, if not greater, in sensitivity to the commercially available, solubilized porcine TSHR assay (6). However, the availability of CHO cells expressing larger numbers of TSHR (26, 27) prompted us to examine whether these cells would provide an even more effective TBI assay. To the contrary, in preliminary experiments, we found that the pig TSHR TBI assay was far superior to an assay using intact CHO cells overexpressing the hTSHR. For example, TBI values (percent inhibition of [125I]TSH binding) with three potent sera were 79%, 91%, and 83% with the commercial assay vs. 10%, 13%, and 57% with the TSHR-10,000 cell line (~2 x 106 receptors/cell), respectively. We, therefore, focused on a comparison of our original cell line (4kb TSHR) with a line (TSHR-0) expressing an intermediate number of receptors per cell (Table 1Go).

A few important points could be made from many experiments that we performed to optimize the intact cell TBI assay. The deleterious effect on TBI sensitivity of a large number of TSHR per cell could be overcome in part by culturing fewer cells with more receptors in smaller wells. Thus, TSHR-0 cells (150,000 TSHR/cell) (26) cultured in microtiter (0.36-cm2) wells provide only about 2-fold more receptors per well than the 4kb TSHR cell line (16,000 TSHR/cell) (7, 27) cultured in 24-well cluster plates (1.77-cm2 wells). The two major advantages of microtiter plates were the use of less serum and a smaller volume of [125I]TSH with less background binding. By this means, samples could be assayed in triplicate, and the tracer concentration could be increased up to 100-fold, with a progressive increase in TSHR saturation (Fig. 1AGo). Nevertheless, TBI values with the TSHR-0 cells were lower than those with the 4kb TSHR cell line, reflecting the greater number of TSHR per well with the former cells (Fig. 1BGo).



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  		Figure 1. A, Specific [125I]TSH binding to CHO cell monolayers expressing the hTSHR on their surface. To obtain similar numbers of TSHR per well, 4kb TSHR cells and TSHR-0 cells were cultured in 24- and 96-well cluster dishes, respectively. Cells were incubated with the indicated concentrations of [125I]bTSH (250 and 50 µL/well in the 24- and 96-well plates, respectively). Specific binding was determined by subtraction of tracer bound to untransfected CHO cells in parallel wells. Data shown are the means for duplicate (24-well) and triplicate (96-well) determinations. B, TBI assay using 4kb TSHR and TSHR-0 cells cultured in 24- and 96-well plates, respectively. The assay was performed with the same Graves’ patient serum at the indicated concentrations of [125I]bTSH. Data shown are the mean ± range of values for duplicate (24-well) and the mean ± SE of triplicate (96-well) determinations.

 
Finally, in these studies of intact hTSHR-expressing CHO cells, we compared the use of [125I]bTSH with [125I]hTSH in a TBI assay using TSHR-0 cells plated in microtiter wells. With sera from 12 patients, covering a wide range of TBI values, the data obtained were generally similar regardless of the species of radiolabeled TSH used (Fig. 2Go). Nevertheless, of interest and potential importance, a few sera (for example, no. 1, 3, 6, and 8) produced greater inhibition of radioligand binding with hTSH than with bTSH. It must be emphasized, however, that the use of hTSH in a TBI assay is presently limited because this species of TSH is a less effective ligand than bTSH (33). Indeed, in the experiment shown, maximal [125I]TSH binding was 5-fold lower with hTSH than with bTSH (4.9% vs. 24.8%).



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  		Figure 2. Comparison of radiolabeled hTSH and bTSH in a TBI assay. Data are shown for 12 sera assayed using TSHR-0 cells (~150,000 TSHR/cell) cultured in a 96-well microtiter plate. The TBI assay is described in Materials and Methods. Fifty microliters of either [125I]bTSH or [125I]hTSH (5 x 104 cpm; 106 cpm/mL) were added to each well. Values shown are the mean ± SE of triplicate wells for each tracer. **, P < 0.001; *, P < 0.01 (by Student’s t test). Tracer TSH binding in the presence of normal serum was: [125I]bTSH, 12,396 cpm (mean of 12,044, 12,323, and 12,821 cpm); hTSH, 2,442 cpm (mean of 2,509, 2,362, and 2,454 cpm).

 
TSH binding inhibition assay using detergent-extracted recombinant hTSHR

As mentioned above, intact CHO cells expressing large numbers of receptors on their surface cannot be used for TBI assays. However, we wished to determine whether such cells would be a good source of recombinant TSHR in a soluble receptor assay. For this purpose, we used cells (TSHR-10,000), that express very large numbers (~1.9 x 106) of TSHR on their surface (27). Cells were suspended by scraping, homogenized in buffer containing 1% Triton X-100, and compared with the solubilized porcine TSHR in the universally used commercial kit as a standard. Recombinant receptor extracted from 7 x 106 cells was required to obtain [125I]bTSH binding comparable to that of the porcine TSHR standard (Fig. 3Go). In contrast to this low yield, far more TSHR capable of TSH binding was recovered when monolayers of the same cells were incubated with detergent-containing buffer without detaching the cells from the culture dishes. In this case, TSHR from 70-fold fewer (~105) cells produced binding similar to that of the porcine TSHR standard. Indeed, a single 10-cm diameter dish of cells provided sufficient TSHR for 100–200 replicate determinations when substituted for the porcine TSHR in the kit.



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  		Figure 3. Scraping and homogenizing TSHR-10,000 cells reduces the yield of TSHR extracted with detergent. TSHR were extracted from cells by two different procedures. First, cells were scraped from 50 confluent 10-cm dishes (5 x 108 cells) and pelleted. After extraction with buffer containing 1% Triton X-100 (see Materials and Methods, aliquots (50 µL) derived from the indicated number of cells was substituted for the same volume of porcine TSHR normally used in the kit. Second, cell monolayers were directly extracted with buffer containing 1% Triton X-100 without removing the cells from the culture dishes (see Materials and Methods). Three milliliters of detergent-containing buffer were added to a 10-cm diameter culture dish (107 cells). During dilution of the cell extracts, the detergent concentration was kept constant. The final concentration of 0.25% Triton X-100 in the assay was found not to affect [125I]TSH binding. Each point represents the mean ± the range of duplicate determinations. The dashed vertical lines indicate the number of CHO cells needed to attain TSH binding equivalent to 50 µL solubilized porcine TSHR, defined as 100% (dashed horizontal line).

 
The efficacies of the solubilized human and porcine TSHR were compared in a TBI assay using sera from 30 individuals with known or suspected Graves’ disease. Ten of these sera had undetectable TSHR autoantibodies using the porcine TSHR in the kit (TBI, <15%). The remaining 20 sera contained a wide range of TSHR autoantibody activity (Fig. 4Go). TBI values obtained when hTSHR was substituted in the kit correlated very well with values determined with porcine TSHR (r = 0.954; P < 0.001). However, two sera that were negative with porcine TSHR antigen were positive with the hTSHR (Fig. 4Go). Radiolabeled hTSH could not be used in the soluble TSHR assay because, unlike with [125I]bTSH, PEG precipitation of the tracer was strongly and variably influenced by the individual sera (data not shown).



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  		Figure 4. Comparison of solubilized porcine and human TSHR in a TBI assay. TBI activity was determined in 30 sera sent to a clinical laboratory for known or suspected Graves’ disease. Sera were tested with a commercial kit using porcine TSHR. In addition, the same sera were assayed with the same reagents, except that solubilized hTSHR was substituted for porcine TSHR (see Materials and Methods). The cut-off point for positivity as defined in the kit (TBI >15%) is indicated. Each point represents the mean of closely agreeing duplicate determinations. The arrow indicates two sera with detectable TBI activity with the hTSHR, but not with the porcine TSHR.

 
TBI values obtained with the solubilized porcine TSHR are known to correlate only weakly with thyroid-stimulating activity determined in a bioassay (TSI) involving activation of the hTSHR (11, 23, 34). We, therefore, wished to determine whether TBI values obtained with solubilized hTSHR correlated better with the bioactivity of IgG in the same sera. Sufficient serum was available from 28 of the 30 samples depicted in Fig. 4Go to permit determination of TSI activity using CHO cells stably transfected with the hTSHR. The correlation between TBI and TSI activities was no better when human, rather than porcine, TSHR was used in the TBI assay (r = 0.732 and 0.709, respectively; Fig. 5Go, A and B).



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  		Figure 5. Correlation between TSI activity and TBI activity determined with solubilized human (A) or porcine (B) TSHR. TSI activity was determined on 28 of the sera depicted in Fig. 4Go using a bioassay involving CHO cells stably transfected with hTSHR (see Materials and Methods). Each point represents the mean of closely agreeing duplicate determinations. The hatched area indicates the mean ± 2 SD of cAMP levels (130% of basal) determined in sera from normal individuals (n = 20).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data in the present study provide important insight into the development of the next generation of assays for the detection of TSHR autoantibodies. First, it is interesting (and perhaps counterintuitive) that mammalian cells expressing more TSHR on their surface provide a less sensitive TBI assay when this assay is performed with intact cells in monolayer culture. The most likely reason for this phenomenon is the very low absolute concentration in serum of TSHR autoantibodies. Although previously suspected, only very recently has it been shown by flow cytometry that TSHR autoantibodies are generally present at much lower concentrations than thyroid peroxidase autoantibodies in the same serum (35). The efficacy of an occupancy assay such as the TBI depends on an excess of antibody over antigen. Thus, the combination of a large amount of antigen (TSHR) and a low concentration of antibody leads to low occupancy of the antigen. This, in turn, will reduce the sensitivity of the TBI assay because many TSHR unoccupied by antibody are available for [125I]TSH binding. In retrospect, we were fortunate that our previous intact cell TBI assay (23) used CHO cells transfected with the 4kb TSHR cDNA. We only realized subsequently that the untranslated regions of the TSHR messenger ribonucleic acid reduce the level of TSHR expression in CHO cells (26). An optimum TBI assay, therefore, should use a small amount of receptor and a very effective ligand, as is the case with the affinity-purified [125I]bTSH in the procedure developed by Rees-Smith (6, 29).

A TBI assay using cell monolayers is at a serious practical disadvantage relative to assays using solubilized TSHR. However, although solubilized porcine TSHR preparations are clearly effective, hTSHR have been suggested to be the most appropriate for study of TSHR autoantibodies (29). The most important contribution of the present study is the demonstration for the first time that soluble recombinant hTSHR can readily be obtained from mammalian cells in large amounts and in a form suitable for an effective TBI assay. Soon after we generated a stably transfected CHO cell line (7), we found that TSH binding to cells scraped from culture dishes was greatly reduced compared to binding to cells in monolayer culture (Rapoport, B., and G. D. Chazenbalk, unpublished observations), hence our initial use of intact cells in a TBI assay (23). Similarly, examination of the data reported by Costagliola et al. reveals that the yield of solubilized TSHR from scraped JP09 cells is much lower than expected (25). The yield of solubilized TSHR from stably transfected mouse myeloma cells grown to high density in a fermentor (10) has not been reported. The present study demonstrates this low recovery of effective TSHR from resuspended cells and indicates that the direct extraction of TSHR from cell monolayers can overcome the evident fragility of this very difficult receptor. However, only a cell line such as TSHR-10,000 that expresses very high levels of TSHR can provide TSHR suitable for direct use in a TBI assay without further purification or concentration.

There is evidence that the TSHR species may be important in bioassays for stimulatory autoantibodies (11, 34, 36). Whether the use of human, rather than porcine, TSHR would be advantageous in a TBI assay remains to be established. This factor was considered during the original development of the TBI assay using solubilized porcine TSHR (29). However, because TBI values in 18 sera did not differ greatly when TSHR of either species was used, and because of the easier access to porcine than to human thyroid tissue, porcine TSHR became the standard in TBI assays. It is worth noting that most sera in this previous study had relatively high TBI values, making discrimination at the very important low end of the assay difficult to discern. The present availability of solubilized recombinant hTSHR allowed us to reassess this question. Consistent with the previous data from nonrecombinant material (29), TBI values of 30 sera were generally comparable with those of solubilized porcine and hTSHR. However, a small proportion of sera was positive using hTSHR, but negative using porcine TSHR. It is presently unclear whether such discrepancies represent false positives or reflect genuine differences in the recognition of human vs. porcine TSHR. These findings reveal the need for a future study, involving a large number of clinically defined patients, on the relative sensitivities of TBI assays using solubilized human and porcine TSHR.

Finally, the question arises as to whether, in addition to TSHR species, the species of the ligand (TSH) used in TBI assays is important. hTSH is a very difficult ligand to use in a TBI assay because of its lower specific activity relative to that of bTSH, even when interacting with the hTSHR. Nevertheless, in a TBI assay using intact cells, hTSH and bTSH ligands did not produce identical results with all sera. The variable background together with low absolute binding precluded us from using hTSH in a solubilized TSHR TBI assay. The reason for the variable background with hTSH is unclear. However, there are previous observations that Igs in Graves’ sera bind hTSH to a greater degree than do Igs from normal individuals (37). The recent development of hTSH superanalogs (38) may permit TBI assays using both hTSHR and hTSH.

In summary, we report that in an intact cell TBI assay, 1) the use of cells with more TSHR reduces, rather than improves, the sensitivity of the assay; and 2) the species of radiolabeled TSH (bovine vs. human) can influence the TBI value obtained. On the other hand, cells expressing very large numbers of receptors are an excellent source of detergent-solubilized TSHR, but only when the extraction procedure is modified. These findings open the way to the development of a practical TBI assay using recombinant hTSHR.


   Acknowledgments
 
We thank the National Hormone and Distribution Program, the NIDDK, the Center for Population Research of the NICHHD, the Agricultural Research Service of the USDA, and the University of Maryland School of Medicine for kindly providing the highly purified bTSH for radioiodination.


   Footnotes
 
1 This work was supported by NIH Grant DK-19289. Back

Received February 4, 1997.

Revised March 20, 1997.

Accepted March 25, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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