0021-972x/92/7506-1540$03.00/0 .Journal of Clinical Endocrinology and Metabolism Copyright 0 1992 by The Endocrine Society

Vol. 75, No. 6 Printed in U.S.A.

Binding Assay for Thyrotropin Receptor Using the Recombinant Receptor Protein* S. COSTAGLIOLA, AND M. LUDGATE

S. SWILLENS,

P. NICCOLI,

J. E. DUMONT,

Autoantibodies

G. VASSART,

Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucleaire (S.C., S.S., J.E.D., M.L.), Universite Libre de Brurelles, Hopital Erasme, B-1070 Brussels, Belgium; Department of Genetics (G. V.), ULB, Hopital Erasme, B-1070 Brussels, Belgium; and U38 INSERMUA 178 CNRS (P.N.), Faculte de Medecine, Marseille, France ABSTRACT We have characterized a transfected Chinese hamster ovary cell line, JPO9, which expresses high levels of the human TSH receptor (TSH-R). Based on a theoretical biological activity for TSH of 40 III/ mg, JPO9 has approximately 90,000 receptors per cell, having a dissociation constant of 1.64 X 10’” mU/L or 1.47 X lo-” mol/L. We have used JPO9 to prepare solubilized TSH-Rs which have formed the basis of a binding assay for thyroid-binding inhibiting immunoglobulins in unfractionated sera. We have compared the JPO9 assay with the TRAK assay (which is based on solubilized porcine

TSH-R) and found a highly positive correlation between the two assays, r = 0.83 P < 0.0001, in 55 sera from patients with autoimmune thyroid disease. JPO9 can be adapted to growth in suspension culture, permitting large scale production. The tracer in the assay is bovine [““IITSH; surprisingly, despite the use of a hTSH-R, hTSH had no effect on the binding of the tracer up to 10” mU/L and only a minor effect at 10” mu/L. (J Clin Endocrinol Metab 75: 1540-1544, 1992)

T

HE TSH receptor is the target of autoantibodies (autoabs) which act as TSH agonists (TSAb) (1, 2) or antagonists (TBAb) (3, 4), the former resulting in hyperthyroidism and the latter the possible cause of hypothyroidism. Currently a number of methods are in use which measure the binding of TBII (thyroid binding inhibiting immunoglobulin) or bioactivity of TSAb/TBAb and which thus play a role in diagnosis. One of the most frequently used is the TRAK assay, in which autoantibodies compete with [‘251]TSH (bovine) for binding to solubilized porcine TSH receptor (TSH-R). In common with many assays it uses a nonhuman antigen (5). The recent cloning and sequencing of the human TSH-R (6, 7) enabled us to express it in eukaryotic cells (8). We have previously shown that stably transfected Chinese hamster ovary (CHO) cells expressing the TSH-R could be used, in place of human thyrocytes, to measure the production of CAMP in response to TSAb and the inhibition of TSHinduced CAMP production by TBAb in a hypotonic bioassay

have established a binding assay for TSAb/TBAb which is performed on unfractionated serum, using solubilized receptors from these cells and compared it with the TRAK assay. The possibility of growing the cells in suspension culture greatly facilitates their large scale production.

(9).

In order to estimate the number of human TSH-Rs per cell, 40,000-g membrane preparations of JP09 were produced and resuspended in solution B (see below). Protein concentrations were determined by the method of Bradford (10). Five to 10 pg highly purified bovine TSH (40 U/mg kindly supplied by Dr. J. G. Pierce, University of California, Los Angeles, CA) were labeled with lZ51 (Amersham, Oxon, UK) by the lactoperoxidase method. Iodination was performed in 0.5 mol/L aceto-acetate, pH 5.6, and the labeled hormone separated from the free radioactivity on a 1.6 X 70 cm sephacryl ZOOHR column (Pharmacia, Piscataway, NJ) equilibrated in 50 mmol/L Tris, pH 7.2, 0.1% BSA, to give a specific activity of 66 pgCi/ J% Bovine [‘251]TSH binding to 10 WLg JP09 membranes was performed for 1 h at room temperature in an eppendorf tube in a total vol of 200 ~1 in 20 mmol/L Tris, 1 mmol/L EDTA, 0.2% BSA, pH 7.4. The reaction was stopped by centrifugation at 13,000 rpm in a minifuge at 4 C, the supernatant aspirated, and the pellet counted in a y-counter. Under

Materials Production

and maintenance

and Methods of JPO9 cell line

CHO cells were cotransfected with a pSVL construct containing the complete coding sequence of the hTSH-R and pSV2 neo and selected by geneticin resistance to produce a mixed population of cells. These were cloned by limiting dilution, and their accumulation of CAMP in response to TSH, forskolin, and a TSAb were measured to select clones expressing high levels of the hTSH-R, as previously described (8). In this study, clone JP09 has been used.

Characterization

In the same study we also showed that TSH/TSAb and TBAb could displace bovine [‘251]TSH from COS cell membranes which express the TSH-R and could thus form the basis of a radio-receptor assay (RRA) for TSH-R autoabs. In the present study we have characterized the number and affinity of the TSH-Rs expressed in a CHO clone, JPO9. We Received March 17, 1992. Address all correspondence and requests for reprints to: Dr. M. Ludgate, Department of Genetics, ULB, Hopital Erasme, route de Lennik 808, B-1070 Brussels, Belgium. *This text presents research results of the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming.

of JPO9 cell line

1540

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RECOMBINANT these conditions equilibrium was reached (data not shown). The binding of the labeled bovine TSH was competed for, in the same conditions, with increasing concentrations of the same unlabeled bovine TSH or thytropar (Armour Pharmaceuticals Co, Phoenix, AZ). The displacement curves were analyzed by nonlinear regression on the basis of a single class of receptor displaying competitive binding. The analysis accounted for the significant difference between free and added ligand concentrations due to the binding. The number of receptors per cell was calculated using the maximum binding value transformed to molarity, and molecular weight and specific bioactivity for TSH of 28,000 and 40 IU/mg, respectively, a reaction vol of 200 ~1, and a quantity of membranes corresponding to 5 x lo5 cells per tube; it assumes that iodinated TSH has the same affinity for the receptor as noniodinated.

Preparation

of solubilized

assay for autoantibody

determinations

The assay was performed in 1.5-m] eppendorf tubes using 150 ~1 solubilized receptors and 50 ~1 serum which were incubated together for 15 min at room temperature. 100 microliters of bovine [‘*“I]TSH (the kind gift of Henning, Berlin, Germany) were added to each tube, and incubation continued at room temperature for 1 h. The reaction was stopped by adding 400 bl cold 50 mmol/L NaCI, 10 mmol/L Tris, pH 7.5, 0.1% BSA followed by 700 ~1 30% polyethylene glycol in 1 mol/L NaCl. The tubes were centrifuged at 13,000 rpm for 5 min in a minifuge at 4 C, the supernatants were completely aspirated, and y-radiation in the pellets measured. Total binding of [‘251]TSH was measured using 50 ~1 of a pool of 10 normal sera and the nonspecific binding determined by adding an excess (IO-30 mU/ml) of unlabeled bovine TSH (Sigma, St Louis, MO). Results are expressed as a percentage of total binding calculated as:

counts

counts in the presence of test serum in the presence of normal pool (total

The nonspecific

binding

was calculated

All determinations assays.

were

performed

bound)

in duplicate

culture

JP09 cells were grown in 200 ml Ham’s F12 supplemented with 20 mmol/L HEPES, pH 7, as the buffering system in 500 ml sterilized Wheaton culture flasks. They were maintained at 37 C and agitated at 100 rpm. The cultures were seeded with varying numbers of cells (3-35 X 104/ml), several aliquots were removed aseptically each day, and the number and viability of cells per milliliter were determined. Solubilized receptors were prepared as described for the monolayer cultures and compared with these in the binding assay.

Results of the JPO9 cell line

The number of receptors expressed per JP09 cell and their dissociation constants (Kds) were computed from displacement curves in which binding of [‘251]TSH (bovine) was competed for with increasing concentrations of cold bovine TSH. It was estimated that the cells harbored about 90,000 receptors (range 77,000-110,000) with a Kd of 1.64 X lo3 mU/L or 1.47 X lOmy mol/L (range 1.53-1.8 X lo3 mU/L or 1.37-1.61 lo-” mol/L) as determined in three separate experiments. A representative displacement curve is shown in Fig. 1. Establishment

of an RRA using solubilized

JPO9 receptors

We attempted to produce a more concentrated and homogeneous TSH-R preparation by solubilizing the crude membrane fraction using nonionic detergents, Triton and NP40, at O.Ol%, O.l%, and 1% and found that 1% Triton was optimal (data not shown). We compared the inhibition CPM 5000

x 100

in at least two

separate

3000

2000

A total of 78 sera were used: 18 were from normal individuals; 2 from hypothyroid patients with idiopathic myxoedema; 53 from hyperthyroid patients with Graves disease, 18 of whom also had ophthalmopathy. Diagnoses were based on standard clinical and laboratory criteria. Patients’ sera were selected to give a wide range of activities in the TRAK RRA for TSH-R autoantibodies. The bioactivity of the samples was tested in a hypotonic bioassay in which CAMP accumulation of CHO cells transfected with the hTSH-R was measured in the absence and presence of 10 mU bovine TSH and 1.5 mg/ml of each immunoglobulin preparation as previously described (9).

with RRA using solubilized

1541

4000 cold TSH pool

Sera used in the study

Comparison

Suspension

TBII

x 100

as:

counts in the presence of 30 mU/ml counts in the presence of normal

FOR

Characterization

JPO9 receptors

Batches of receptors were prepared from 10 confluent 9-cm petri dishes (a total of -10” cells) as previously described (8). The 40,000-g membrane pellet was resuspended in 1 ml 75 mmol/L Tris, pH 7.5, 12.5 mmol/L MgQ, 0.6 mmol/L EDTA, 1 mmol/L EGTA, 250 mmol/L sucrose containing 1 pmol/L leupeptine, and 1 mmol/L phenylmethylsulfonyl fluoride (solution B) and then added to 4 ml 1% Triton, 50 mmol/L NaCl, 10 mmol/L Tris, pH 7.5, in which it was homogenized. The suspension was centrifuged at 100,000 X g for 2 h at 4 C, and the resulting supernatant was divided into aliquots and stored at -80 C.

Binding

ASSAY

porcine

1000

1

0 1, IO'

TSH-R

All sera samples were analyzed in a TRAK assay (Henning Berlin, Germany) performed according to the manufacturer’s tions.

I IO2

I

I

I

I

IO3

IO4

1 o5

IO6

bTSH (mlU/L) GmbH, instruc-

FIG. 1. Representative tween “‘I-labeled Pierce; 0 thytropar.

displacement curves for the competition and unlabeled bovine TSH to JPO9 membranes; Each point is the mean of two measurements.

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be0

COSTAGLIOLA of bovine [‘251]TSH binding of one normal and three different TSAb containing sera when using the crude membranes and the solubilized preparation (with polyethylene glycol precipitation). We also measured the displaceable TSH binding activity remaining in the membrane fraction after solubilization. As shown in Table 1, similar results were obtained with the crude membranes and solubilized receptors. Furthermore, the majority of the TSH-Rs are solubilized by the method employed, the difference in the total bound to the two preparations probably being due to incomplete solubilization. Finally, before testing a large number of normal and patients’ sera, we investigated the possible interference of human TSH in the assay by measuring the displacement of [““I]TSH by a normal serum and three TSAb containing sera in the presence of increasing doses of hTSH (Boehringer Mannheim, Mannheim, Germany). As shown in Table 2, it had no effect at lo3 mU/L and only a minor effect if any at lo4 mu/L. Similar results were obtained with a highly purified TSH preparation (Therapeutic Products Laboratory CAMR, Salsbury, UK) and when using solubilized JP09 receptors or a crude membrane preparation (results not shown). To eliminate the possible inactivation of TSH during purification, we measured the total binding of bovine [‘25I] TSH (Henning) to JP09 membranes in the presence of normal sera or sera from four nonautoimmune hypothyroid patients with very high levels (176 to >lOOO mu/L) of circulating TSH (the kind gift of J. B. Vanderpas, Hopital Ambroise Pare, Mons, Belgium). There was no difference in the binding observed, despite the hypothyroid sera being capable of stimulating CAMP accumulation in intact JP09 cells (results not shown). Comparison

with an RRA using solubilized

porcine

TSH-R

The results of inhibition of binding of bovine [‘251]TSH to solubilized porcine TSH-R and solubilized recombinant hTSH-R of the 78 unfractionated sera described in Materials and Methods are shown in Fig. 2. There is a significant positive correlation between the two assays, r = 0.8386, P < 0.0001. The counts per min obtained in a typical assay are shown in Table 3. TABLE 1. Inhibition of bovine solubilization using 1% Triton

[‘*“I]TSH

binding

to crude

Crude

Total

bound (2628,

Cold TSH

(30 mu) (842,

Normal

serum (2450,

TSAbl (2425, TSAb2 (1814, TSAb3 (1460, Experiments

were performed

using

40 pg protein

and solubilized

ET

JCE & M. 1992 Vol75.No6

AL.

Suspension

culture

Larger scale production of the JP09 cell line would be facilitated by suspension culture. We were able to adapt the cells to growth in suspension achieving a doubling time of approximately 24 h and a maximum density of about 0.6 X lo6 cells/ml depending on the number of cells per ml in the initial inoculum, the cells continued to express a functional TSH-R (data not shown). We found that cultures were not viable when seeding at less than 5 x lo4 cells/ml. Discussion The availability of the complementary DNA for the human TSH-R makes it possible to express it on a background devoid of other thyroid-specific proteins, permitting the measurement of autoantibodies directed solely against the receptor, thus achieving a greater specificity both for binding and bioassays. In previous studies we provided preliminary characterization of a series of CHO cell lines stably transfected with the hTSH-R complementary DNA (8) and documented the use of one of them (JP26) in a bioassay of TSAb (9). The present study makes use of another of these cell lines, JP09, which is being distributed as a tool to measure TSAb and TBAb. Based on a number of assumptions (see below) JP09 expresses about 90,000 receptors per cell and compares well with the number of receptors achieved by others with an amplifiable vector system (11). It is still about an order of magnitude lower than in the cells described by Chazenbalk et al. (12) (but see below). Interestingly, both our CHO lines of the JP series and that of Harfst et al. (11) display a much higher stimulation of CAMP accumulation in response to TSH (up to ZOO-fold) or TSAb than those described by Chazenbalk et al. (12) (up to ZO-fold only). This discrepancy is probably due to differences in the characteristics of the CHO cells before transfection (e.g. number or type of GTPbinding proteins expressed). The Kd computed for the receptor as expressed in JP09 is about 1.64 X lo3 mU/L or 1.47 X 1 Om9mol/L and compares with that found by others (13, 14), although it is different to the value in our earlier study (8) probably due to the use of impure TSH (Thytropar) in our previous displacement experiments. We have demonstrated that when adding the same quantities of thytropar and Pierce JPO9 membranes

by normal

JPO9 membranes

Solubilized

2600 2572) 780 717) 2496 2542) 2418 2411) 1794 1774) 1430 1400)

1980 (1962,2008) 610 (595,625) 1920 (1933, 1907) (97%) 1841 (1811, 1871) (93%) 1584 (1603, 1565) (80%) 1128

eq/tube

(96%) (93%) (69%) (55%) in each case. Results

JPO9

sera and TSAb;

1%

triton

(1111,1145)

are mean

efficiency

Membranes after 1% Triton

600 (541, 659) 450 (459,437)

(57%)

counts

per min (range).

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of

RECOMBINANT TABLE 2. Effect of human R (solubilized) RRA

TSH

on the human

Normal Serum

Total

bound

TSAbl

(99.5%) 1046 (976,1116)

(98.6%) 1058

1 mU hTSH/ml

(1041,1076)

Results

(1084,1082) (102%)

(99.8%) 1020 (983, 1058) (96.2%)

10 mU hTSH/ml

are mean JPO9

TSAbZ

950 (977,926) (89.6%) 976 (957, 995) (92%) 1082

1055 (982,1128)

0.1 mU hTSH/ml

recombinant

966 (922, 1011) (91%)

of duplicate

counts

TSH-

TSABB

674 (665,683) (63.5%) 654 (713,595) (61.6%) 682 (738,626) (64.3%) 585

370 (390,352)

(547,623)

‘$344’

(34.9%) 360 (356,

387)

(33.9%) 355 (322,387) (33.4%) 330

.

(55%)

per min

ASSAY

0

(range).

ASSAY

l-201

0' 0

10

20

30

40

50

60

TRAK

70

60

90

100

110 120

ASSAY

2. Comparison of inhibition of binding of bovine [‘251]TSH (TRAK) by normal sera, 0, n = 18; TSAb, 0, n = 53; TBAb, Cl, n = 2, and 30 mu/ml bovine TSH (thytropar) (*) to solubilized JPO9 and porcine receptors. Results are expressed as a percentage.

FIG.

TABLE

3. Examples

of counts

per min

Solubilized

Total bound” XS cold TSH Normal serum TBAb TSAbl TSAb2 TSAb3 TSAb4

2842

JPO9

(2847,2837)

610 (581, 638) 2546

(2493,260O)

581 (632,530) 2205 2027

(2263,2147) (2021,2033)

1464 (1463,1465) 766

(711,822)

in a typical Solubilized

assay porcine

3267 (3233,3302) 627 (609, 656) 3320(3365,3275) 634 (640,628) 2504 (2560,2448) 2481(2449,2514) 2036(2067,2005) 1127 (1095,116O)

Total counts per min = 7000. a Performed in the presence of 50 pi/tube normal pool described in Materials and Methods. Results are mean (range).

sera

as

TSH, in terms of bioactivity, the displacement curves are not superposable, indicating that thytropar contains additional TSH molecular species which compete with the tracer in binding experiments. This poses the problem of comparing results from various laboratories for both Kd and number of

FOR TBII

1543

receptors per cell, and, more fundamentally, of determining the true values for these parameters. With figures for the bioactivity of pure TSH ranging from 40-200 U/mg or more (15), it seems futile to express Kds in molar terms, and to attempt obtaining a definite number of receptors per cell. The results presented here make use in displacement experiments of TSH reported originally to have an activity of 40 U/mg (16). In addition, it must be kept in mind that all these experiments make use of bovine TSH. The estimated Kds cannot be extrapolated easily to the human hormone (see below). Despite these theoretical considerations, JP09 cells constitute an ideal source of reagents to measure TSAbs and TBAbs. The fact that the cells can be grown continuously in suspension while keeping their characteristics adds to their usefulness and may provide a powerful means to screen for monoclonal antibodies raised against the receptor by, fluorescence-activated cell sorting. A direct comparison of an assay based on the solubilized human TSH receptor from JP09 with the TRAK assay (based on a porcine receptor preparation), demonstrates a very good correlation (r = 0.83, P < 0.0001). The fact that 5-10% of sera from Graves’ patients score as negative in both assays (less than 10% displacement) suggests that activation of the receptor by some antibodies might involve sites distinct from those where TSH binds, or, less likely but not excluded, that autoantigens other than the TSH receptor might be implicated in stimulation of the adenylyl cyclase. The serum of one (hypothyroid) patient reported to activate CAMP accumulation in FRTL-5 cells and not in COS cells transfected with the hTSH-R might contain autoabs recognizing such an antigen (17). However, the greater sensitivity of FRTL-5 cells for CAMP determinations as compared to transiently transfected COS cells may provide an explanation. In a similar approach, using CHO cells expressing the hTSH-R, Filetti et al. have devised an assay in which immunoglobulin preparations compete with labeled TSH for binding to whole cells. Such a protocol, although perfectly adequate for research purposes, seems cumbersome for routine TBII determinations. A problem to address when measuring TBIIs in unfractionated serum is that of a possible effect of circulating TSH in the competition for [‘251]TSH binding. On a theoretical basis, one might expect that this could be even more prevalent when the human receptor is used. Surprisingly, no significant effect was observed when hTSH (two different preparations) was added to the assay up to 1 O4 mu/L. Under similar conditions and for similar concentrations, bovine TSH displaced the [‘251]TSH tracer completely. This is not due to a reduced bioactivity of the hTSH, since it induced CAMP production to the same extent as the bovine TSH of Pierce and thytropar (19), as did native TSH, present in the circulation of nonautoimmune hypothyroid individuals, which also did not compete with bovine [‘251]TSH for binding to the JP09 membranes. It is difficult to accept the idea that human and bovine TSH interact with different portions of the TSH-R. It has been shown that bovine TSH displays a much higher affinity for the human receptor than hTSH does and that optimum

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COSTAGLIOLA

1544

conditions for the binding of each are similar (20). Recent experiments by Foti et al. (21) have shown that bovine and hTSH binding are differently affected by trypsin treatment of thyroid membrane receptor preparations (21). Although in these experiments the trypsin-treated receptor binds preferentially hTSH, it is conceivable that partial proteolysis which seems to occur during the preparation of receptor for binding studies (22) is responsible for some loss of affinity of the receptor for hTSH. Whatever the reason for these observations, our experiments demonstrate that the assay, as it stands, is unaffected by the TSH concentrations which can be found in patients’ serum. From previous experiments we know that the JP09-CHO cell line constitutes a convenient tool to measure the bioactivity of TSAb/TBAb in CAMP accumulation assays. The present study demonstrates that it is a convenient source of hTSH receptor for assaying TBII in unfractionated serum. Acknowledgments We are grateful to Dr. P. Carayon (Marseille), Dr. J. Orgiazzi (Lyon), Dr. S. Mariotti (Piss), Dr. J, Mockel (Brussels), and Dr. J. B. Vanderpas for providing sera and to Jason Perret for performing the initial transfection. We greatly appreciate the provision of [‘?]TSH and TRAK kits from Henning.

References 1. Rees Smith B, McLachlan J. 1988 Autoantibodies to the thyrotropin receptor. Endocr Rev. 9:106-120. 2. Manley SW, Knight A, Adams DD. 1982 The thyrotropin receptor. Springer Semin Immunopathol. 5:413-431. ’ 1 3. Orniazzi I, Williams DE, Choara II, Solomon DH. 1976 Human thyyoid a&nylyl cyclase &imulating-activity in immunoglobulin G of patients with Graves’ disease. J Clin Endocrinol Metab. 42:341354. 4. Matsuura N, Yamada Y, Nohata Y, et al. 1980 Familial neonatal hypothyroidism due to maternal TSH-binding inhibiting immunoalobulins. N Enal I Med. 303:738-741. K, C;hagh FM, Teece M, Kingswood C, Ress-Smith B. 5. gouthgate 1984 A receptor assay for the measurement of TSH receptor antibodies in unextracted serum. Clin Endocrinol. 20:539-543. 6. Libert F, Lefort A, Girard C, et al. 1989 Cloning, sequencing and expression of the human thyrotropin (TSH) receptor: evidence for binding of autoantibodies. Biochem Biophys Res Commun. 165: 1250-1255. 7. Nagayama Y, Kaufman K, Seto P, Rapoport B. 1989 Molecular cloning sequence and functional expression of the cDNA for the human thyrotropin receptor. Biochem Biophys Res Commun.

ET AL.

JCE & M. 1992 Vol75.No6

165:1184-1190. 8 Perret J, Ludgate M, Libert F, et al. 1990 Stable expression of the human TSH receptor in CHO cells and characterization of differentially expressing clones. Biochem Biophys Res Commun. 171: 1044-1050. 9. Ludgate M, Perret J, Gkrard C, et al. 1990 Use of the recombinant human thyrotropin.receptor expressed in mammalian cell lines to assay TSH-R autoantibodies. Mol Cell Endocrinol. 73:R13-R18. 10. Bradford MM. 1976 Rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding. Anal Biochem. 72:248-254. 11. Harfst E, Johnstone Al’, Gout I, Taylor AH, Waterfield MD, Nussey SS. 1992 The use of the amplifiable high-expression vector pEE14 to study the interactions of autoantibodies with recombinant human thyrotropin receptor. 12. Chazenbalk GD, Nagayama Y, Kaufman KD, Rapoport B. 1990 The functional expression of recombinant human thyrotropin receptors in nonthyroidal eucaryotic cells provides evidence that homologous desensitization to thyrotropin stimulation requires a cellspecific factor. J Clin Endocrinol Metab. 127:1240-1244. 13. Carayon P, Guibout M, Lissitzky S. 1979 The interaction of radioiodinated thyrotropin with human plasma membranes from normal and diseased thyroid gland. Ann Endocrinol. 4O:Zll. 14. Pekonen F, Weintraub BD. 1980 Salt induced exposure of high affinity receptors on human and porcine thyroid membranes. J Biol Chem. 255:8121. 15. Rapoport B, Seto P. 1985 Bovine thyrotropin has a specific bioactivity 5- to lo-fold that of previous estimates for highly purified hormone. Endocrinology 116:1379-1382. 16. Pierce JG. 1974 CheGistry of thyroid stimulating hormone. In: Greeu RO, Astwood EB, eds. Handbook of Phvsiologv. Washington “’ DC: American Physiological Society; vol 2:79.’ 17. Endo T, Haraguchi K, Ohmori M, Ikeda M, Ohta K, Onaya T. 1991 Thyrotropin receptor non-mediated thyroid stimulating immunoglobulin in Graves’ disease. Biochem Biophys Res Commun. 179:1543-1547. 18. Filetti S, Foti D, Costante G, Rapoport B. 1991 Recombinant human thyrotropin (TSH) receptor in a radioreceptor assay for the measurement of TSH receptor autoantibodies. 1 Clin Endocrinol Metab. 72:1096-1101. 1 19. Laurent E, Van Sande J, Ludgate M, et al. 1991 Unlike thyrotropin, thyroid stimulating antibodies do not activate phospholipase C in human thyroid slices. J Clin Invest. 87:1634-1642. J, Thompson B, Edmonds C. 1981 Comparison of binding 20. Kermode of bovine and human thyroid-stimulating hormone to receptor sites on human thyroid membranes. J Endocrinol. 88:205-217. 21. Foti D, Russo D, Costante G, Filetti S. 1991 The biological activity of bovine and human thyrotropin is differently affected by trypsin treatment of human thyroid cells: thyroid stimulating antibody is related to human thyrotropin. J Clin Endocrinol Metab. 73:710-716. 22. Russo D, Chazenbalk GD, Nagayama Y, Wadsworth HL, Seto I’, Rapoport B. 1991 A new structural model for the thyrotropin (TSH) receptor, as determined by covalent cross-linking of TSH to the recombinant receptor in intact cells: evidence for a single polypeptide chain. Mol Endocrinol. 5:1607-1612.

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