105

Biochem. J. (1975) 145, 105-111 Printed in Great Britain

A Radio-Ligand Receptor Assay for the Long-Acting Thyroid Stimulator INHIBITION BY THE LONG-ACTING THYROID STIMULATOR OF THE BINDING OF RADIOIODINATED THYROID-STIMULATING HORMONE TO HUMAN THYROID MEMBRANES By S. QASIM MEHDI* and STEPHEN S. NUSSEY Nuffield Department of Clinical Biochemistry, University of Oxford, Radcliffe Infirmary, Oxford OX2 6HE, U.K. (Received 7 May 1974) Highly purified bovine TSH (thyroid-stimulating hormone)

was

labelled with 125I by

using very low concentrations of chloramine-T. Human thyroid membranes prepared by discontinuous sucrose-density-gradient centrifugation were homogeneous on examination by electron microscopy. Incubation of radioiodinated TSH with the membranes showed that radioactivity could be bound to the membranes. Under the experimental conditions described here, binding was dependent on time and temperature and was a saturable phenomenon. Preincubation of the membranes with unlabelled hormone inhibited the subsequent binding of 125I-labelled TSH. Similarly, inhibition by the long-acting thyroid stimulator also showed a saturation behaviour. A rapid and sensitive method for the detection of the long-acting thyroid stimulator is described. LATS,t usually detected by bioassay of its mouse thyroid stimulatory activity (McKenzie, 1958), is found in the IgG fraction of the serum proteins of only some patients with Graves' disease (McKenzie, 1968). The apparent inability to demonstrate the presence of this stimulator in sera of all such patients has been attributed to causes which include the insensitivity of the bioassay (Wong & Litman, 1969) and to the presence of an IgG akin to LATS, which is able to react with, and stimulate, human thyroid activity but not that ofthe mouse (Adams & Kennedy, 1967). According to the theory of Sutherland et al. (1968), the physiological stimulator of the thyroid, TSH, acts by first binding to a specific receptor on the thyroid plasma membrane (Pastan et al., 1966). This binding stimulates adenylate cyclase activity (Yamashita & Field, 1970) and the resulting increase in the intracellular concentrations of cyclic AMP activates various thyroidal enzyme systems. LATS also stimulates adenylate cyclase activity (Bastomsky & McKenzie, 1968). Although TSH and LATS are physicochemically and immunologically different, the effects of the two stimulators on thyroid function in a number of mammals is qualitatively the same. Hence it is possible that both TSH and LATS may act at the same site on the thyroid plasma membrane. * Present address: Klinikum Steglitz der Freie Universitat Berlin, 1 Berlin 45, Hindenburgdamm 30, West Germany. t Abbreviations: LATS, long-acting thyroid stimulator; IgG, immunoglobulin G; TSH, thyroid-stimulating hormone (thyrotrophin).

Vol. 145

Previous work from this and other laboratories has supported the view that like TSH, LATS acts on the cell membrane (Mehdi et al., 1971). The present work stems from a detailed study on the binding of TSH to human thyroid membranes (S. Q. Mehdi & S. S. Nussey, unpublished work). The data presented here describe some aspects of the binding of 125I-labelled TSH to human thyroid membranes and the inhibition of this binding by unlabelled TSH or LATS. A rapid and sensitive method for the detection of LATS in human sera (or IgG prepared from them) is described in detail. A brief, preliminary report of some of these findings has been published (Mehdi et al., 1973).

Materials and Methods Sera and IgG Sera were from normal euthyroid individuals and from patients presenting at the outpatient clinics of the Radcliffe Infirmary, Oxford, and the Klinikum Steglitz, Berlin. All sera were tested for LATS activity by the mouse bioassay method described by McKenzie & Williamson (1966). IgG was obtained from sera by Na2SO4 fractionation (Kekwick, 1940) followed by chromatography on DEAE-Sephadex A-50 by the method of Baumstark et al. (1964). Radioiodination of TSH Highly purified bovine TSH (30-40i.u./mg) was a gift from Professor J. G. Pierce (UCLA School of

106 Medicine, Los Angeles, Calif., U.S.A.). lodination by a modification of the method of Hunter & Greenwood (1964). For this I.OmCi of 125I (Na'251, IMS 30; The Radiochemical Centre, Amersham, Bucks., U.K.) was added to 2.5,ug of TSH in 105p1 of 0.5M-sodium phosphate buffer, pH7.5. Then 5,ug of freshly dissolved chloramine-T in 5p1 of the buffer was added and after min at room temperature (23-25°C) the reaction was stopped by the addition of 15,pg of freshly dissolved sodium metabisulphite in 5,ul of the buffer. Two drops of a transfer solution [IO% (w/v) sucrose, 1 % KI and 0.01 % Bromophenol Blue] were added and the mixture was chromatographed on a column (1 cm x 15cm) of Sephadex G-50 (fine grade). The column was previously washed with Sml of 5 % (w/v) bovine serum albumin in the elution buffer [0.05M-sodium phosphate buffer (pH7.5)0.1 M-NaCI], and fractions (0.5ml) were collected in an equal volume of the buffer solution containing bovine serum albumin. Radioactivity was measured by using Packard or Wallac auto-gamma counters. Specific radioactivities of 30-75pCi/4ug were obtained on different occasions. Iodinated TSH showed no significant loss of biological activity in the McKenzie & Williamson (1966) bioassay. 1251-labelled TSH was stored in refrigeration at 0-20C and used within 1 week of iodination. was

Preparation of thyroid homogenates and plasma membranes Human thyroids were obtained at operation or post-mortem and kept on ice until frozen within 15min of removal. The tissue was dissected free of fibrous material, cut into slices with a razor blade and homogenized with a Silverson mixer-emulsifier (six 5s exposures) in 3 vol. of 0.01 M-sodium phosphate buffer, pH7.5, containing 0.01 M-MgCI2 plus 0.25 M-sucrose. All operations were at 0-40C. The suspension was then homogenized with four to six strokes of a hand-operated Potter-Elvehjem homogenizer with a loose-fitting Teflon pestle. The homogenate was centrifuged at lOOOg for 10min. The pellet was resuspended in a small volume of the same buffer, homogenized and centrifuged. This procedure was repeated three times to wash the fibrous pellet. Membranes were obtained from the combined supernatants (referred to as lOOOg-supernatant) initially by discontinuous sucrose-density-gradient centrifugation. A lOml portion of the lOOOg supernatant was made 1.31 M with respect to sucrose and layered over 5ml of the homogenization buffer containing 2.0M-sucrose in a centrifuge tube. Two over-layers of lOml and 5ml of the buffer contained 1.23M- and 0.8M-sucrose respectively. After centrifugation for 12h at 25000rev./min in a Beckman model L2-65B ultracentrifuge in rotor SW 25.1,

S. Q. MEHDI AND S. S. NUSSEY material at the 2.OM-/1.31M-, 1.31M-/1.23M- and 1.23M-/0.8M-sucrose interfaces was removed into separate tubes by aspiration with a Pasteur pipette. The pellet at the bottom of the tube was also resuspended in a small volume of buffer. The above four samples were diluted with phosphate-buffered saline containing bicarbonate (0.01 M-sodium phosphate buffer, pH7.5, 0.14M-NaCl and 0.01 M-NaHCO3) to decrease the viscosity caused by the presence of sucrose and centrifuged at 105OO0g for 90min. The pellets so obtained were resuspended in the same buffer with a few gentle strokes of a hand-operated Potter-Elvehjem homogenizer and washed twice by centrifugation and resuspension. The final pellets were resuspended in a small volume of the phosphatebuffered saline solution containing bicarbonate. Any coarse material was removed by centrifugation at lOOOOg for 10min. The above fractions were tested for their ability to adsorb LATS activity as described by Beall & Solomon (1966) and to bind 125I-labelled TSH as described below. For this, each fraction [0.4mg of protein, measured by the method of Itzhaki & Gill (1964)] was incubated with 125I-labelled TSH (approx. 25000c.p.m.; dilutions were made with phosphatebuffered saline containing bicarbonate) in a final volume of 0.6ml in cellulose nitrate or polyallomer centrifuge tubes. After 6h at 25°C, the tubes were centrifuged at 105000g for 90min in a Spinco rotor 40.3. The supernatant was carefully aspirated with a Pasteur pipette and the pellet was obtained by cutting out the bottom of the tube. Radioactivities in the pellet (bound '25I-labelled TSH) and supematant (free 125I-labelled TSH) fractions were measured for each sample. For all subsequent experiments membranes were prepared by carefully layering a sample (35-45 ml) of the lOOOg supematant over 15-25 ml of 0.01 Msodium phosphate buffer containing 0.01 M-MgCI2 and 1.31M-sucrose in a Spinco SW 25.2 tube. After centrifugation for 4h at 25000rev./min, membranes forming a white suspension at the interface of 1.31 M-sucrose with the lOOOg supernatant sample were harvested by aspiration with a Pasteur pipette. Then 3-4vol. of ice-cold phosphate-buffered saline containing bicarbonate was added to dilute the sucrose and the membranes were pelleted by centrifugation at 105000g for 1 h. The membrane pellet was washed twice and finally resuspended in a small volume of the same buffer. Membranes were stored as concentrates in small batches at -200C. Before use, they were thawed and any aggregates removed by centrifugation. Membrane protein was measured as described above and the suspension diluted to the desired membrane concentration with phosphatebuffered saline containing bicarbonate. Freezethawing had no effect on the binding of l25l-labelled TSH. Membrane concentrates could be stored for 1975

The Biochemical Journal, Vol. 145, No. 1

Plate

1

EXPLANATION OF PLATE I Electron micrograph ofpurified human thyroid membranes (magnification x32400)

S. 0. MEHDI AND S. S. NUSSEY

(Facing p. 106)

The Biochemical Journal, Vol. 145, No. 1

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EXPLANATION OF PLATE 2 Electron micrograph ofpurified human thyroid membranes (magnification x58 500)

S. Q. MEHDI AND S. S. NUSSEY

Plate 2

RADIO-LIGAND RECEPTOR ASSAY FOR THYROID STIMULATORS several weeks in the frozen state. Electron micrographs (kindly provided by Mr. D. W. Jerome of the Nuffield Department of Clinical Pathology) showed that membranes were free of subcellular organelles though occasionally a few microsomes could be seen (Plate 1). Measurement of 125I-labelled TSH binding to human thyroid membranes Membranes (0.2ml; 0.4-1.0mg of protein) in phosphate-buffered saline-bicarbonate solution were transferred to centrifuge tubes which were kept on ice and contained 0.4ml of the same buffer [or buffer containing unlabelled TSH (Thytropar; Armour Pharmaceutical Co., Phoenix, Ariz., U.S.A.)]. Then 20,u1l of 125I-labelled TSH (20000-50000c.p.m.) was added and the tubes were incubated for 3h at 25°C, unless stated otherwise for the individual experiment. At the end of the incubation, 1 ml of ice-cold phosphate-buffered saline-bicarbonate solution was added and the tubes were centrifuged at 105 OOOg for I h. Control samples contained membranes which were preincubated with 0.5i.u. of unlabelled TSH for 3h before the addition of label. These samples provided values for non-specific uptake, which was approx. 2 % of the total radioactivity added. Pellet and supernatant radioactivities were measured as described above. Attempts to separate membrane-bound hormone from the free hormone by polyethylene glycol treatment or by filtration through Millipore filters proved unsuccessful.

Standard procedure for the assay of LA TS in human sera or IgG Human thyroid membranes (0.4mg of protein) in 0.4ml of phosphate-buffered saline solution containing bicarbonate were preincubated in centrifuge tubes with 0.2ml of serum (or IgG solution). A second tube for each sample contained excess (0.5i.u.) of unlabelled TSH for measuring non-specific uptake. For convenience, incubations were carried out at room temperature (23-25°C). After 3 h, 20,u1 of 125I-labelled TSH (approx. 20000c.p.m.) was added with a micropipette and the tubes were incubated for a further 3 h. Then 1 ml of the ice-cold buffer solution was added and the tubes were centrifuged at 105000g for 1 h. Radioactivities in the pellet and supernatant were measured as described above. Samples containing sera (or purified IgG) of normal euthyroid individuals (two or three samples in duplicates) were included as controls. The standard incubation protocol given above could be altered if desired. Preincubation of thyroid membranes with test samples at 0-40C for 6-9h or at 37°C for 1 h, followed by incubation with 1251-labelled Vol. 145

107

TSH for the same period, gave results that were comparable with those obtained by the standard

procedure. Instead of purified membranes, a crude membrane preparation obtained from the 10OOg supernatant by centrifugation at 25000g for 30min was also used for binding studies. This material often gave very high non-specific uptake values, making interpretation of results extremely difficult. Bioassay of LATS activity The mouse bioassay was performed as described by McKenzie & Williamson (1966). Results are expressed as: Blood radioactivity at 12h Initial blood radioactivity

Results and Discussion Beall & Solomon (1966) observed that incubation of LATS serum (or IgG) with whole thyroid homogenates decreased the mouse thyroid stimulatory activity of the serum (or IgG). The four fractions obtained from human thyroid homogenates, by discontinuous sucrose-density-gradient centrifugation, were tested for their ability to adsorb LATS activity and to bind 125I-labelled TSH. Fig. 1(a) shows that the greatest enrichment of 125I-labelled TSHbinding material was in the fraction at the interface of 1.23M-/0.8M-sucrose. LATS adsorption by each of these fractions (Fig. lb) was comparable with their ability to bind 125I-labelled TSH. Finally, material unable to enter 1.31 M-sucrose accounted for 90% of the adenylate cyclase activity ofthe looog supernatant (results not shown). These observations suggested that the thyroid membranes had the ability to bind I'5I-labelled TSH and to adsorb LATS activity. Some characteristics and requirements for TSH binding to human thyroid membranes which are pertinent to the present study are described briefly below.

TSH binding to human thyroid membranes The binding of radioiodinated TSH to human thyroid membranes in phosphate-buffered salinebicarbonate solution is both time- and temperaturedependent (Fig. 2). Binding was also proportional to the concentration of membrane protein (Fig. 3). Similarly, incubation of a constant quantity of membranes with increasing amounts of TSH showed that binding increased proportionately until saturation was achieved (Fig. 4). Preincubation of membranes with unlabelled hormone inhibited the binding of 125I-labelled TSH (Fig. 5). No such inhibition was observed after the incubation of membranes with the other hormones

108

S. Q. MEHDI AND S. S. NUSSEY

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0

20

40

60

80

Decrease in bioassay

binding [bound/free (%.)] response (Y.) Fig. 1. A comparison ofthe ability offractions obtainedfrc9m human thyroid homogenates to (a) bind 125I-labelled TS;H and (b) adsorb LATS activity The four fractions obtained from thyroid homogenates by discontinuous sucrose-density-gradient centrifugation in a Spinco rotor SW 25.1 at 25000rev./min for 12h at 4°C were (I) pellet sedimenting in 2.0M-sucrose and membranes from the interfaces of (II) 2.0M-/1.31 M-sucrose, (III) 1.31 M-/1.23M-sucrose and (IV) 1.23M-/0.8M-sucrose. (a) Some 0.4mg of protein of each of the four fractions was incubated with 125I-labelled TSH and the binding was measured by the procedure described in the Materials and Methods section; (b) LATS adsorption was by incubating portions (5 ml) of a LATS serum [McKenzie & Williamson (1966) bioassay response at 12h was 1024±104] with 5mg of protein/ml from each of the four samples. After incubation for h at 37°C, tubes were centrifuged at 105 OOOg for h to remove particulate material and the supernatant (serum) was tested for LATS activity in the McKenzie & Williamson (1966) bioassay.

that were tested [human growth hormone, luteinizing hormone, prolactin and adrenocorticotrophic hormone (Synacthen, CIBA Laboratories, Horsham, Sussex, U.K.); S. Q. Mehdi & S. S. Nussey, unpublished work]. The inability of these hormones to inhibit the specific binding of '25I-labelled TSH, saturation of binding sites on the membranes with TSH (Fig. 4) and the inhibition of binding in the presence of excess of unlabelled TSH show that binding was to specific receptors on the thyroid membranes.

Effect of LATS on TSH binding to human thyroid membranes Yamashita & Field (1972) and Kendall-Taylor (1973) showed that LATS stimulated thyroidal

1

3

2

5

4

6

Time (h) Fig. 2. Effect of time and temperature on the binding of 125l-labelled TSH to human thyroid membranes Membrane (0.4mg of protein) in 0.6ml of phosphatebuffered saline solution containing bicarbonate was incubated with 130pg of 1251-labelled TSH (approx. 30000c.p.m.) at 0-40C (@), 25°C (A) and 35°C (u). Results have been corrected for non-specific uptake.

80-

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3

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Membrane protein (mg/ml) Fig. 3. Binding of TSH to increasing amounts of thyroid membranes The experimental procedure was the same as in Fig. 2 except that 540pg of 1251-labelled TSH (approx. 45000c.p.m.) was incubated at 25°C for 4h with the amount of membrane proteins indicated.

adenylate cyclase activity in plasma membranes. If both TSH and LATS react with the same receptors on the thyroid membrane, then LATS, like unlabelled TSH, should be able to inhibit the specific binding of radioiodinated TSH (Fig. 5). The effect of both LATS serum and LATS IgG on the binding of '25l-labelled TSH was examined. Fig. 6 shows that preincubation of membranes with serum inhibited the subsequent binding of '25I-labelled TSH and that the inhibition was proportional to the amount of LATS serum in the sample. 1975

RADIO-LIGAND RECEPTOR ASSAY FOR THYROID STIMULATORS

200 d)

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1.'0

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TSH added (munits/tube) Filg. 4. Binding of TSH to thyroid membranes as a function of hormone concentration Assay conditions were the same as in Fig. 3. All tubes contained the same amount of '251-labelled TSH (2ng = 40,uunits = 100000c.p.m.) and unlabelled TSH was added to give the concentrations indicated on the abscissa. Bound TSH was calculated by making appropriate corrections for the specific activity in each sample.

20

0

40

80

60

100

LATS serum cY.) Fig. 6. Effect of preincubation of membranes with LATS serum on the binding of'25I-labelled TSH LATS serum [McKenzie & Williamson (1966) bioassay response at 12h was 1195±139] was diluted with normal (LATS-negative, euthyroid) serum. Assay was under standard conditions.

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Unlabelled TSH added (munits/tube) Fig. 5. Effect ofpreincubation ofmembranes with unlabelled TSH on the binding of 125I-labelled TSH Membranes were preincubated with increasing concentrations of unlabelled hormone as indicated and the subsequent binding of 1251-labelled TSH was measured under standard conditions.

With LATS IgG it was possible to examine the effects of LATS concentration over a wide range (Fig. 7). IgG was prepared from 50ml of a LATS serum and resuspended in 5 ml of phosphate-buffered saline solution containing bicarbonate (theoretically a tenfold concentration). Appropriate dilutions were made with the same buffer solution and the samples were assayed by the standard procedure described in the Materials and Methods section. It is clear that inhibition of 125I-labelled TSH binding to membranes caused by the presence of LATS also showed saturation behaviour. Vol. 145

x

0. I x

l.Ox

10x

[LATS-IgG]

m

Fig. 7. Effects of various concentrations of LATS IgG on the binding of TSH IgG was prepared from 50ml of a LATS serum [McKenzie & Williamson (1966) bioassay response at 12h was 1239±150] and resuspended in 5ml of phosphate-buffered saline-bicarbonate solution. This (lOx) concentrate contained 80mg of IgG/ml. Dilutions were with the same buffer solution and assays by the standard procedure.

It is interesting that after solubilization of thyroid membranes with Triton X-100 or Lubrol-PX, LATS inhibition of the binding of 125I-labelled TSH to solubilized receptors could still be elicited (Mehdi et al., 1974). Thus, although it cannot be claimed that both TSH and LATS react with the same receptors on the thyroid membranes, our results suggest that if different sites are involved they are intimately related. The advantage of the radio-ligand receptor assay described in the present paper is that LATS in human serum (or IgG) is detected by its effects on the binding of TSH to human thyroid membranes. Therefore all LATS and LATS-like activity (Adams & Kennedy, 1967) is expected to be detectable in this system. The

S. Q. MEHDI AND S. S. NUSSEY

110 Patients initials

(a) t

(b)

+

CONTROL A U.L.B. A.C.

M.H.

FE K.U.H. IR CONTROL B L.L. E Ftt

CONTROL C

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We are grateful to Mr. J. R. P. O'Brien for his encouragement and intcrest in this work and to Dr. G. K. Radda for reading this manuscript and for much valuable advice. Thanks are due to Mr. A. S. Till, F.R.C.S., for human thyroid tissue and to Professor H. Schleusener and Dr. T. D. R. Hockaday for sera. S. S. N. is a recipient of an M.R.C. Scholarship for Training in Research Methods. This work was partly supported by an Award of the Wellcome Trust to S. Q. M.

CONTROL D S.CH. R.W. CONTROL E

T.O.W.

1600

800

0

Bioassay response (12h)

100

50

0

LATS inhibition of TSH binding (%o of control) 0i

2

4

Bound/free (%) Fig. 8. Comparison ofLATS activitv in sera by the McKenzie & Williamson (1966) bioassay (a) and the radio-ligand receptor assay (b) For further details see the text.

8

(a)

results of an assay are given in Fig. 8. For comparison, McKenzie & Williamson (1966) bioassay values are included for each sample. IgG concentrates (fourfold or sevenfold) of sera from several patients with thyrotoxicosis (Graves' disease), that were LATSnegative in the bioassay, failed to inhibit TSH binding to membranes (Fig. 9). Unless an additional or alternative mechanism of thyroid stimulation by LATS (other than by effects on. the receptor-adenylate cyclase system) is proposed, our results do not support the view that LATS or a LATS-like humanspecific thyroid-stimulating IgG (Adams & Kennedy, 1967; also see Doniach, 1973) is continuously present in the sera of all patients suffering from Graves' disease.

References Adams, D. D. & Kennedy, T. H. (1967)J. Clin. Endocrinol. Metab. 27, 173-177 Bastomsky, C. & McKenzie, J. M. (1968) Endocrinology 83, 309-314 Baumstark, J. S., Laffin, R. J. & Bardawill, W. A. (1964) Arch. Biochem. Biophys. 108, 514-522 Beall, G. N. & Solomon, D. H. (1966) J. Clin. Endocrinol. Metab. 26, 1382-1388

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Fourfold concentrates Sevenfold concentrates Fig. 9. Effects ofIgG concentrates from thyrotoxic LATS-negative sera on the binding of'25I-labelled TSH to human thyroid membranes All sera were from patients suffering from Graves' disease. McKenzie & Williamson (1966) bioassay responses at 12h were less than 200. (a) Fourfold (approx. 32mg of IgG/ml) and (b) sevenfold (approx. 55mg of IgG/ml) IgG concentrates were used for assay by the standard procedure. Control (unshaded) and LATS-positive IgG (horizontal bars) are included for comparison.

1975

RADIO-LIGAND RECEPTOR ASSAY FOR THYROID STIMULATORS Doniach, D. (1973) Symp. Advan. Med. 9th (Walker, G., ed.), pp. 27-37, Pitman Medical, London Hunter, W. M. & Greenwood, F. C. (1964) Biochem. J. 91,43-56 Itzhaki, R. F. & Gill, D. M. (1964) Anal. Biochem. 9, 401-410 Kekwick, R. A. (1940) Biochem. J. 34, 1248-1257 Kendall-Taylor, P. (1973) Brit. Med. J. 3, 72-75 McKenzie, J. M. (1958) Endocrinology 63, 372-382 McKenzie, J. M. (1968) Physiol. Rev. 48, 252-340 McKenzie, J. M. & Williamson, A. (1966) J. Clin. Endocrinol. Metab. 26, 518-526 Mehdi, S. Q., Hockaday, T. D. R., Newlands, E. & ElKabir, D. J. (1971) Proc. Roy. Soc. Med. 64, 12681269

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Mehdi, S. Q., Nussey, S. S., Gibbons, C. P. & ElKabir, D. J. (1973) Biochem. Soc. Trans. 1, 10051006 Mehdi, S. Q., Nussey, S. S, Simpson, R. D. & Adlkofer, F. (1974) Endocrinol. Exp. 8, 163-164 Pastan, I., Roth, J. & Macchia, V. (1966) Proc. Nat. Acad. Sci. U.S. 56, 1802-1809 Sutherland, E. W., Robison, G. A. & Butcher, R. W. (1968) Circulation 37, 279-306 Wong, T. W. & Litman, G. W. (1969) J. Clin. Endocrinol. Metab. 29, 72-78 Yamashita, K. & Field, J. B. (1970) Biochem. Biophys. Res. Commun. 40, 171-178 Yamashita, K. & Field, J. B. (1972) J. Clin. Invest. 51, 463-472

A radio-ligand receptor assay for the long-acting thyroid stimulator. Inhibition by the long-acting thyroid stimulator of the binding of radioiodinated thyroid-stimulating hormone to human thyroid membranes.

Highly purified bovine TSH (thyroid-stimulating hormone) was labelled with 125I by using very low concentrations of chloramine-T. Human thyroid membra...
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