Endocrinology Copyright 0 1992 by The Endocrine Society

Vol. 130, No. 2 Printed

Characterization of Monoclonal Thyrotropin Receptor S. MARION,





in U.S.A.

to the Human


INSERM U.283, HGpital Cochin (S.M., A.R., J.M.B., J.C.), 75674Paris Cedex 14, France; and the Universitt?Libre de Bruxelles (M.L.), Brussels,Belgium

ABSTRACT. We have produced four monoclonal antibodies (mAbs), 34A, 49G, llE7, and 12E3, which bind the human TSH receptor (hTSH-R) when expressed on a human thyroid cell line (GEJ). freshlv dissociated human and murine thyroid cells, or Chinese hamster ovary cells stably transfected with the hTSHR gene. These mAbs were obtained after immunization of DBA/ 1 mice with affinity-purified TSH-binding sites from GEJ cells. Biochemical studies, including sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, Western blot, and immunopreciuitation of solubilized GEJ cell membranes or human thvroid cells showed that most of the mAbs recognized two bands: one located at 46-48 kilodaltons and the other at 86-86 kilodaltons. Inhibition of [‘251]hTSH binding to solubilized porcine membranes (TSH-receptor auto-antikorper assay) or Chinese ham-


UMEROUS experiments have suggested that immunoglobulins (Ig) in the serum of patients with Graves’ disease (GD) have a pathogenic role and cause thyroid gland hyperactivity. In this hypothesis, thyroid gland overactivity is the result of the TSH receptor (TSH-R) being stimulated by the patient’s own IgG, mimicking the action of the hormone TSH (for review, see Ref. 1). Data favoring this hypothesis include inhibition by GD’s Ig of [1251]TSH binding to thyroid cells, measurement of CAMP production, or iodide uptake when thyroid cells are incubated with GD’s Ig (2). As well as the thyroid-stimulating antibodies of GD (TSAb), patients with myxoedema may have thyroid-blocking antibodies (TBAb), which result in hypothyroidism. The target of TSH, TSAb, and TBAb is the TSH-R, and we have recently established a human thyroid hybridoma (GEJ) that has enabled us to solubilize, purify, and characterize the TSH-R of these cells. In the meantime, the cloning and sequencing of the TSH-R have been reported in a number of species (3-6). There is no doubt that antibodies to the TSH-R, both mono- and polyclonal, will be useful tools to increase our underReceived July 24, 1991. Address all correspondence and requests for reprints to: J. Charreire, INSERM U.283, Hopital Cochin, 27 rue du Fg St. Jacques, 75674 Paris Cedex 14, France.

ster ovary cell membranes previously transfected with hTSH-R gene showed that mAb 34A recognizes the hTSH-binding site of both receptors. In contrast, mAbs 49G, llE7, and 12E3 recognize a structure located near the hTSH-binding site. Lastly, the ability of these mAbs to stimulate murine thyroid function was investigated by measuring CAMP production and iodide accumulation. The 34A mAb, which fullv comnetes with [‘251]TSH for binding to hTSH-R; was able-to induce both functions. Conversely, the 12E3 mAb, which was the least potent inhibitor of [‘251]TSH binding to hTSH-R-transfected cells had no effect. A relationship was, therefore, established between the capacity of mAb to hTSH-R to inhibit [‘251]hTSH binding and their ability to induce thyroid functions. (Endocrinology 130: 967-975, 1992)

standing of the modes of action of TSAb and TBAb, and while awaiting the large scale expression of the recombinant TSH-R, a number of groups have produced antibodies to synthetic peptides of the TSH-R (7,8). Data exist which suggest that autoantibodies will bind only to the native TSH-R (9). For this reason, we have produced monoclonal antibodies (mAb) in mice by immunizing them with TSH affinity-purified material from GEJ cells. We report the characterization of four such mAb. Materials

and Methods

hTSH (NIDDK hTSH B.l) was supplied by the National Pituitary Agency (Baltimore, MD). Its biopotency was 15 IU/ mg in terms of WHO hTSH 68/38, determined by the McKenzie mouse method. For studies of binding to Chinese hamster ovary (CHO) cell membranes, unlabeled bovine TSH (Thytropar, Armour Co., Kankakee, IL) and [““IITSH, a gift from Henning GmBH (Berlin, Germany), were used. In the RRA, we used the TSH-receptor auto-antikorper (TRAK) assay kit, kindly provided by Henning. BSA (fraction V), potassium iodide, Triton X-114 (Tx114), trichloroacetic acid, 3-isobutyll-methylxanthine, phenylmethylsulfonylfluoride (PMSF), pepstatin, antipepsin, benzamidine, insulin, Pansorbin, hypoxanthine, azaserine, and thymidine were obtained from Sigma (St. Louis, MO). Cyanogen bromide (CNBr)-activated Sepharose 4B, protein-A, and relative molecular mass (Mr) standards


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were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). CAMP accumulation was evaluated using the [“‘I] CAMP assaysystem (reference no. RPA 508) from Amersham (Les Ulis, France). Nitrocellulose filters were obtained from Schleicher and Schuell (Dassel,Germany). Collagenasedispase was purchased from Boehringer (Mannheim, Germany), RPMI-1640 was obtained from Gibco (Paisley, Scotland, United Kingdom), and polyethylene glycol (PEG) 1000 was obtained from Merck (Darmstadt, Germany). Microtiter plates (reference no. 3590) and 96- or 24-well culture plates were obtained from Costar (Cambridge,MA). ““I was purchased from CEA (Saclay, France). Human thyroid peroxidase was a gift from Dr. P. Carayon (Marseille, France), and human thyroglobulin (Tg) from Dr M. Schlumberger (Villejuif, France). Rabbit antimouse (RAM) (Nordic) Ig, phosphatase-and peroxidase-labeledgoat antirabbit (GAR Ig PA; Nordic and Biosys, Compiegne, France), and fluorescein-conjugated goat antimouseIg (GAM Ig) were obtained from Cappel (West Chester, PA). Peroxidase-labeledGAM Ig waspurchasedfrom Biosys. Vectors pSVL and pSV, NE0 were obtained from Pharmacia and Dr. Martial (Liege, Belgium) respectively. Immunization

of animals

Six- to 8-week-old female DBA/l (H-2”) mice were used. They were injected SCinto four or five siteswith approximately 5 pg affinity-purified TSH-binding sites from GEJ cells after emulsion in complete or incomplete Freund’s adjuvant. The hTSH-R preparations contained, after electrophoresisand silver staining, two major bands; one is specific for hTSH and located at 48-50 kilodaltons (kDa), whereas a secondis nonspecific and located at 67 kDa. Moreover, a minor band is located at approximately 90 kDa, as previously described(10). However, before emulsification, antigenic preparations were transferred onto nitrocellulose, as describedby Knudsen (11). The immunization schedulewas one monthly injection for 8 months, with animals bled 7 days after each injection. The binding to affinity-purified hTSH-R was measured in each immune serum. Cells thyroid hybridoma (GEJ) (12). Cells were maintained in complete culture medium RPMI-1640, 100 U/ml penicillin, 100 pg/ml streptomycin, and 7.5% heat-inactivated fetal calf serum (FCS) in an atmosphereof 10% CO, in air. GM 1500 6TG A12, the hybridization partner of human thyroid cells, were grown under the sameconditions and used as negative control cells. Human

thyroid cells.These were obtained from human paronodal tissue from thyroids removed becauseof GD or benign nodules.Tissue sampleswere dissectedand minced at 4 C in RPMI-1640 culture medium. The suspensionwas incubated with collagenasedispaseat 5 mg/ml in a shaking water bath for 30 min at 37 C. The cells were then washedin PBS before use. Human

CHO cells.CHO cellswere cotransfected with the entire coding sequenceof the hTSH-R reported by one of us (4) in the shuttle vector pSVL and pSV, NEO. GeneticinStably transfected

Endo. 1992 Vol130.No2

resistant cells were cloned by limiting dilution, and of the 24 clones resulting, JPO9 was selectedas expressing the highest level of hTSH-R, as measuredby CAMP accumulation in responseto TSH (13). The cells were routinely grown in 25-cm2 culture flasks in Ham’s F-12 medium supplementedwith 1 mM sodiumpyruvate, 100U penicillin, 100pg streptomycin, 2.5 pg/ ml amphotericin-B, and 10% FCS and maintained in a 5% CO1, water-saturated incubator at 37 C. Membrane preparations and hTSH-R purifications were performed as previously described(7). Briefly, freshly dissociated human thyroid or GEJ cells resuspendedin PBS containing 0.5% Tx114 and various proteaseinhibitors (PMSF, iodoacetamide,antipepsin, pepstatin, and benzamidine) were incubated on ice for 30 min and then centrifuged (8000rpm; 8 min; 4 C). The supernatant was incubated for 5 min at 33 C to allow separation of the detergent and aqueousphases.After a centrifugation (8 min; 8000 rpm; 22 C), the supernatant was removed, and the detergent-rich phase was retreated before dilution in ice-cold PBS supplemented with 5 mM protein inhibitors to obtain 0.1% Tx114. The suspensionwas then depositedovernight at 8 C on a hTSH Sepharosegel preparedpreviously. Briefly, 400pg hTSH were covalently linked to 1 mg CNBR-activated Sepharose4B usingthe procedurerecommendedby the supplier. After extensive washingswith a mixture of PBS (pH 7.5), 1 M NaCl, 0.05% Tx114, and protease inhibitors, the bound material was eluted with 0.1 N glycine-HCl buffer, pH 2.3. Membrane preparations of stably transfected CHO cellswere prepared as previously described (13). Briefly, cells were harvested by scraping the dishers with a rubber policeman after replacing the mediumwith PBS (without Mg”’ and Ca’+). After low speed centrifugation, the cell pellet was resuspendedin buffer containing 15 mM Tris-HCl (pH 7.5), 2 mM MgC&, 0.3 mM EDTA, 1 mM EGTA, 1 mM PMSF, and 5 pM leupeptin and subjectedto two freeze-thaw cyclesin liquid nitrogen. Cells were further ruptured by passingthrough a glasshomogeneizer. The suspensionwas centrifuged 30 min at 40,000x g, and the crude membranepellet wasresuspendedin buffer [75 mM TrisHCl (pH 7.5), 125 mM MgCl?, 0.3 mM EDTA, and 250 mM sucrose]. An aliquot was assayedto determine protein content, using the Bradford assay(14). Cell fusion and cloning

Immune spleencells from one mousewith serumexhibiting binding to affinity-purified hTSH-R were usedand fusedwith the nonsecreting hypoxanthine-guanine-phosphoritosyltransferase-deficient BALB/c myeloma P3X63 (8653) according to the method of Kohler and Milstein (15). Briefly, spleencells in RPMI-1640 were mixed with myeloma cells at a 1O:l ratio. Fusion wasperformed using40% PEG 1000.Thereafter, fusion cultures in RPMI-1640 supplemented with 15% FCS were seededin 96-well microtiter plates. On day 8 after seeding, selective hypoxanthine, azaserine,and thymidine mediumand, later, hypoxanthine-thymidine medium were added. From day 25, the wells containing cells under vigorous growth were harvested, and their supernatantswere screenedfor anti-hTSH-R antibody production using the enzyme-linked immunosorbent assay(ELISA) method, as describedabove. Cells from positive wells were transferred into 24-well plates and cloned twice by

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mAbs TO THE hTSH-R limiting dilution (0.4 cell/well). Then, mAb was purified from mouse ascitic fluid by chromatography on a protein-A-Sepharose CL4B (Pharmacia Fine Chemicals, Piscataway, NJ). Affinity


Five milligrams of each mAb were fixed to 1 ml CNBractivated Sepharose4B gel. Then, solubilized GEJ cell membranes or human thyroid cells from 5 x 10’ cells were loaded onto each affinity chromatography column. Bound proteins eluted with HCl-glycine buffer (pH 2.3; 0.1 M) were dialyzed, analyzed by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and silver stained. Binding

studies of mAb to various species of R-TSH

A method identical to that used for fiadrenergicreceptorswasemployed(16). Affinity-purified TSHbinding sites (-20 rig/well) from GEJ cells were fixed to wells of microtiter plates with 50% methanol. The wells were saturated with 0.2 ml 1% BSA in PBS for 1 h at 22 C, rinsed three times with 0.05% BSA-PBS, and incubated for 90 min at room temperature with 0.05 ml of each clone supernatant or mAb at the specified concentration in 1% BSA-PBS. The wells were then washed 10 times with 0.05% BSA-PBS and incubated sequentially with 0.05 ml RAM Ig diluted 1:250for 2 h at 22 C and 0.05 ml phosphatase-labeledGAR Ig, asthe third antibody, diluted 1:lOO in 1% BSA-PBS. The wells were washedextensively with distilled water to remove unbound third antibody, and 0.05 ml substrate solution (1 mg/ml; P-nitrophenylphosphate in 10% diethanolamine and 0.5 mM MgCl,) was added. The plates were incubated at 22 C for 2 h, and absorbancewas read at 405 nm. ELISA wasalsousedfor the detection of crossreactivity of mAb to hTSH-R with thyroid antigens such as thyroglobulin and thyroid peroxidase.

Solid phase ELISA.

analysis. GEJ or human thyroid cells (5 x lo”) werewashedtwice (200 x g; 10 min) in PBS-l% BSA, fixed in 1% paraformaldehyde in PBS, and incubated (1 h; 37 C) with 50 ~1 mAb appropriately diluted. Cells were washed twice in cold PBS, incubated with the fluorescein-conjugatedGAM Ig (50 ~1; 1:lOO; 1 h at 4 C), washedtwice, resuspendedin 400 ~1 PBS, and analyzed by the FACSCAN analyser (Becton Dickinson, Mountain View, CA). Control sampleswere similarly incubated with 1 unrelated murine mAb and the secondstep reagent. The percentage of positive cells was determined over 5000 events acquired. Logarithmic means were counted and comparedto those of controls.

Flow cytometry

TRAK assay.One to 200 rg/ml mAb in 50 ~1were addedto 50 ~1 detergent-solubilized porcine TSH-R for 15 min at room temperature. Then, 100 ~1 [‘?]bovine TSH were distributed into each sampleand incubated for 60 min at 37 C before the addition of 2 ml cold PEG and centrifugation (2000 X g; 20 min; 0 C). Lastly, the supernatant wasdiscarded,and the pellet radioactivity was counted in a y-scintillation counter (LKB, Bromma, Sweden). Results are expressedas inhibition of labeled TSH binding determined by the following formula in which nonspecific binding (NSB) is defined as counts in the absenceof detergent-solubilizedporcine TSH-R: 100 X [( [lnnI] TSH specifically bound in the presenceof mAb - NSB)/( [““I]


TSH specifically bound in the absenceof any competitor NSB. Significant receptor activity wasconsideredto be present if inhibition of TSH binding exceeded10%. All analyseswere carried out in triplicate in three separateassays. [‘251]TSH saturation and competitive binding assay to hTSHR-transfected CHO cells.Assayswere performed in 1.5-ml tubes

on ice in a total volume of 200 ~1;the buffer was 20 mM TrisHCl (pH 7.5), 4 mM MgCl, and 0.5 mM BSA. Twenty-five micrograms of protein from membranes of JPO9 cells were incubated for 15 min with 50 ~1mAb (500 kg/ml dilution) with agitation at room temperature. One hundred microliters of [“‘I] TSH were added to each sample,which was incubated for a further hour in the sameconditions. The tubes werecentrifuged for 15 min in a Minifuge, and all the supernatants were removed. The radioactivity remaining in the pellet wasmeasured in a y-counter and compared with the counts when the mAb was replaced by an unrelated mAb; nonspecific binding was measuredin the presenceof an excessof cold TSH. All measurements were made in at least duplicate. Duplicate values agreeto at least 10% and usually to 5%. Results are expressed as: (counts in the presence of experimental mAb - NSB)/ (counts in the absenceof any competitor - NSB) x 100, in which NSB is defined as: (counts in the presenceof XS cold TSH)/(counts in the absenceof any competitor). Western blot

Solubilized membranesfrom GEJ cellswere electrophoresed, aspreviously described(lo), and proteins weretransferred onto nitrocellulose filters previously saturated with nonfat milk and incubated with experimental or control mAb (35 pg/ml in 5 ml for 18 h at 4 C) before sequential addition of RAM antibody (1:250; 1 h at 37 C), peroxidase-labeledGAR antibody (1:lOO; 1 h at 37 C), and enzyme substrate. Between the addition of each reagent, extensive washingswith PBS were performed. Immunoprecipitation

of hTSH-R

Cell surface radiolabeling with leiI was performed by the lactoperoxidase method, as we previously described(7). After labeling, lo7 cells werewashed3 times in RPMI-1640 and lysed by 0.1% TxlOO or 0.5% Tx114; 1 mM EDTA, EGTA, and PMSF; 0.5 pg/ml leupeptin; antipapain; pepstatin; and 50 mM iodoacetamidein 300 ~1PBS on ice for 30 min. After 2 rounds of preclearing (60 min; 4 C) with 100 ~1Pansorbin and 100 ~1 control mAb coupled to Sepharosebeads,the cell lysate was divided into aliquots and incubated for 2 h with experimental mAb coupled to Sepharosebeads.Beads were then washed5 times with lysis buffer and oncewith the samemediumwithout detergent,boiled 10 min in the presenceof Laemmli (17) sample buffer, and centrifuged. The supernatant was analyzed on a 10% SDS-PAGE. CAMP accumulation

Fifty microliters of collagenase-dissociated murine thyroid cells (one lobe per tube) were washedin Hanks’ Balanced Salt Solution containing 0.5 mM isobutylmethylxanthine before the addition of 200 rg/ml mAb in 50 ~1and incubation for 90 min at 37 C. Then, 500 ~1 ethanol were added for 18 h, disrupted cells were stored at -20 C before centrifugation (200 x g; 15

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Endo. 1992 Vol 130. No 2

Characteristics of mAb to hTSH-R

min; 4 C), and the ethanolic extracts were evaporated to dryness. Residues were then assayed for intra- and extracellular CAMP evaluations, performed as recommended by the supplier of the [““I]cAMP assay system. Briefly, samples were reconstituted in 200 ~1 0.05 M acetate buffer with 0.01% NaNrj. To 100 ~1 from this solution, 100 ~1 [““I]cAMP and 100 ~1 rabbit anti-CAMP serum were added for a 4-h incubation at 4 C. Lastly, antirabbit serum coupled to magnetic beads was added for 10 min at 20 C and centrifuged for 20 min (200 x g) to separate the antibody-binding fraction. The radioactivity present in each tube was counted in a y-scintillation counter.


Iodide uptake studies

Culturemedium Valuesare the mean+ triplicate.

Aliquots of 2 x 10” collagenase-dissociatedmurine thyroid cells were incubated for 60 min at 37 C with various amounts of mAb and 0.5 &i “‘1. Then, 0.65 ml 0.05 mg/ml potassium iodide and 0.2 mg/ml BSA mixture were added under Vortex to stop the reaction, and 1 ml of a 20% trichloroacetic acid solution was added before centrifugation (400 x g; 10 min; 4 C). This procedure was repeated twice, and each time the supernatant was discarded.Lastly, pellets were counted in a yscintillation counter.


mAb tested

Bindingto” Isotvne 1TPO


34A 49G llE7 12E3 lE4 5E12 12BlO 12Bll


+ + + -c NT 70 +21 + 37 + 75 + 61 77 40 38

6 10 3 5

47 + 51 + 30 f 20 + 127 + 41 + 46 + 18 f 31+

6 3 7 12

hTSH-R* 4 10 8 20 53 6 4 12 15

408 347 362 189 236 234 357 289 94

+ + -c f + + f + +

70 48 120 24 26 29 94 50 24

of three or four determinationsin

’ Tested in ELISA (absorbance at 405 nm). * Solubilized affinity-purified hTSH-R from ’ Not tested.

GEJ cells.

Results Selection of mAb to hTSH-R

Cloned hybridomas resulting from the fusion of spleen cells from one DBA/l mouse immunized with solubilized affinity-purified hTSH-R from GEJ cells were rapidly screened using a ELISA in which 20 ng affinity-purified hTSH-R from GEJ cells were plated. Of eight mAb detected, seven reacted only with hTSH-R and not with other thyroid antigens, such as thyroglobulin or thyroid peroxidase. Moreover, we demonstrated that they do not react with hTSH (data not shown). In this work, four mAb were studied because of their easy growth and good level of mAb production; they were named 34A, 49G, llE7, and 12E3. We showed that their heavy chains were IgG2a for 34A and 49G, IgGl for llE7, and IgG2b for 12E3. They all had K-light chains (Table 1).



30 mAb(pglml)




1. Binding, determined by ELISA, of anti-hTSH-R mAb to afiinity-purified hTSH-R from GEJ cells. Shown are the mean + SEM of three experiments. n , 34A; 0,49G; 0, llE7; 0, 12E3; A, control mAb. FIG.


so1 60 -

Binding of mAb to hTSH-R To affinity-purified hTSH-R. GEJ cells were solubilized in Tx114 and passed through a hTSH Sepharose column

before acidic elution and dialysis. Then, hTSH-R were coated to plastic plates for measurement of binding of mAbs in the ELISA. Selected mAb and one control mAb were used in concentrations ranging from 1.5-70 pg/ml. As shown in Fig. 1, dose-effect responses were observed, with optimal binding to purified hTSH-R at 35 pg/ml. Considering the hierarchy of the binding to hTSH-R, 34A exhibited the highest binding capacity; 12E3, 49G, and llE7 showed decreasing capacities. To intact GEJ cells and human thyroid cells. Binding of

mAb was also studied using intact

cells bearing








mAb(pg/ml) FIG. 2. Flow cytometric 5000 cells were analyzed.

analysis of mAb binding W, 34A; 0,49G; 0, llE7;

to intact GEJ cells; 0, 12E3; A, control


unmodified TSH-R, such as GEJ cells or human thyroid cells. For that purpose, flow cytometry analysis was performed on cells that were incubated with 6-100 pg/ ml control mAb or mAb to hTSH-R. As shown in Fig. 2, each mAb to hTSH-R specifically bound to the GEJ cells; furthermore, 80-90% maximum binding was reached at a concentration of 30 pg/ml mAb. However, in contrast to the binding observed to purified hTSH-R

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mAbs TO THE hTSH-R from GEJ cells, mAb 12E3 exhibited a higher binding capacity than the other mAbs, suggesting that this mAb recognized an unmodified structure on hTSH-R. When the same experiment was conducted using freshly dissociated fixed human thyroid cells (Fig. 3), only mAb to hTSH-R were able to bind to human thyroid cells. Moreover, mAb exhibit a hierarchy of binding roughly similar to that in intact GEJ cells. To porcine thyroid membranes. The TRAK receptor assay was used to assess the capacity of mAb to recognize the hormone-binding site on the porcine TSH-R. As shown in Fig. 4, this experiment was conducted using four concentrations of each mAb (1,50,100, and 200 pg/ ml). Of the four mAbs, one, 49G, behaved similarly to the control mAb and never competed with [‘251]bovine TSH for binding to TSH;R. Two others, llE7 and 12E3, partially competed (10% specific inhibition); moreover, this competition was detected only when the highest amount of mAb was used. In contrast, mAb 34A competed very effectively with [1251]bovine TSH for binding to TSH-R, since at the four concentrations tested, ap-


proximately 50%, 55%, 70%, and 95% inhibition TSH binding was observed.

of [1251]

To transfected CHO cells. The mAb were tested for their ability to inhibit the binding of [lz51]TSH to membranes prepared from JPO9 cells, in which the only thyroidspecific protein expressed is the hTSH-R and which have been previously shown to bind [‘“51]TSH with a Kd of 50 PM (13). The results are shown in Table 2. The four mAbs to hTSH-R inhibited [1251]TSH binding to CHO cells from 99.6% to 52.1% (Table 2A), whereas it was 28.6% in the presence of a control mAb. It must be noted that 34A totally inhibited (99.6%) [‘251]TSH binding to CHO cells, while 12E3 partially inhibited the binding (52.1%). This specificity of 34A mAb for the hTSHbinding site was further reinforced in dose-effect experiments (Table 2B); when 34A was used diluted 2.5 or 5 times, potent inhibition of [1251]TSH binding was still observed. Affinity


hTSH, mAb 34A or 12E3, or an unrelated mAb was covalently bound to CNBr-Sepharose to obtain four different affinity columns. Subsequently, solubilized GEJ cell membranes provided by 5 x lo8 cells were loaded onto each affinity column. As shown in Fig. 5A, acidic elutions of proteins from the ligand and mAb affinity columns were electrophoresed under reducing conditions TABLE 2. Inhibition of binding of [‘251]hTSH to recombinant R expressed on CHO cell membranes by mAbs to hTSH-R Condition

FIG. 3. Flow cytometric analysis of mAb binding (100 pg/ml) to intact human thyroid cells; 5000 cells were analyzed. a, Control mAb; b, 12E3 (45.5%); c, llE7 (27.0%); d, 49G (21.8%); e, 34A (11.5%).



5 l-


: II 5


c0 E s L5



30 0


100 mAb




FIG. 4. Displacement of binding of mAbs to hTSH-R to solubilized bovine thyroid membranes by [““I]bovine TSH (mean f SEM of three determinations). n , 34A; 0, 49G; 0, llE7; 0, 12E3; A, control mAb.

Exp A No inhibitor XS TSH (30 mu/ml) 34A (50 P1) 49G (50 /LB 12E3 (50 /.d) llE7 (50 ~1) Mixture of the 4 mAbs (50rl) Control mAb (50 ~1) Exp B: dose effect No inhibitor 34A 50 /.ll 20 /.ll 10 /.kl Mixture of the 4 mAb 50 /.Ll 20 pl 10 Wl

Counts (mean i sm) 5037+116 54lk 32 556+16 1968 + 149 2693 zk 75 2086k 93 1055 + 60

% Bound


Inhibition” of binding 6)

100 10.5* 11 39 53 41 19

99.6 68.2 52.1 65.6 88.5




1607 f. 4


267 + 5 382+ 20 958+ 16

16 24 59

84 76 41

312 + 6 661 f 30 937 + 107

19 41 58

81 59 42

All mAbs were used at 500 rg/ml. a Inhibition of binding was calculated as: (experimental binding NSB)/(maximum binding (in the absence of inhibitor) - NSB] X 100. * NSB.

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Endo Voll30.











1992 No 2


86 86

67 48


FIG. 5. Affinity chromatographic purification and SDS-PAGE analysis under reducing conditions and silver staining of solubilized GEJ membranes (A) or human thyroid cells (B). For affinity chromatography, the following ligands were bound to Sepharose 4B: in A: lane 1, mAb 34A, lane 2, mAb 12E3; lane 3, hTSH; and lane 4, control mAb, in B: lane 1, mixture of the four mAbs.

and silver stained. The acidic elution provided by the unrelated mAb affinity column only detected a nonspecific band located at 67 kDa (lane 4). Lane 3 represents the acidic elution of the solubilized membrane proteins from the hTSH column, where proteins of approximately 48-50 kDa Mr were eluted. Lanes 1 and 2 give the acidic elutions of solubilized membranes from anti-hTSH-R mAb columns 34A and 12E3, respectively. Bands located at approximately 48 kDa were observed; for mAb 34A, a weak band located at approximately 86 kDa was also detected. In a second series of experiments, an affinity column was constructed by covalent binding to CNBR-Sepharose of a mixture of the four mAbs (in equivalent amounts). Solubilized membranes from freshly dissociated human thyroid cells were applied to the column, and bound proteins were eluted, as reported above, before SDS-PAGE analysis and silver staining. As shown in Fig. 5B, the acidic elution containing products that had bound to the mixture of the four mAbs showed two bands located at approximately 48 and 87 kDa; the 48-kDa band was the more intense. Western blot analysis of the GEJ cell hTSH-R SDS-PAGE under reducing conditions of solubilized membranes from 10 x lo6 GEJ cells revealed after fast green staining several bands of proteins (Fig. 6, lane 2). To characterize the proteins to which the mAb bound, immunoblot analysis was performed. mAb 34A (lane 3), 49G (lane 4), and 12E3 (lane 5) bound exclusively to proteins located at approximately 53 and 88 kDa; no binding was observed to control cells (lane 7). However, the intensity of mAb 34A binding to proteins of approximately 88 kDa was higher. It must also be noted that only mAb to hTSH-R were able to bind to proteins from GEJ cells; no binding was observed with the control mAb (lane 6).


FIG. 6. Western blot analysis: solubilized GEJ cell membranes (10 x lo6 cells/lane) were electrophoresed under reducing conditions, transferred onto a nitrocellulose membrane that was exposed to mAb to hTSH-R. Lane 1, Mr standards; lane 2, fast green staining. Transferred proteins were exposed to mAb 34A (lane 3), mAb 49G (lane 4), mAb 12E3 (lane 5), or control mAb (lane 6). In lane 7 is a mixture of the three mAbs on control nonthyroid cells.

( k”D’-, 1 200







92 69


_ -84



FIG. 7. Immunoprecipitation under nonreducing conditions of solubilized ‘251-labeled GEJ cell membranes by control mAb (lane l), 34A (lane 2), 49G (lane 3), llE7 (lane 4), and 12E3 (lane 5).

Immunoprecipitation GEJ cells

of ‘251-labeled membranes from

The purified mAb 34A, 49G, llE7, and 12E3; one control mAb; and hTSH were used to precipitate specifically ‘251-labeled membrane-associated proteins from 10 x lo6 GEJ cells. All mAb were used at 4 pg, except 12E3, which was used at 20 pg. The precipitated proteins were separated by SDS-PAGE and analyzed by autoradiograph. As shown in Fig. 7, mAb 34A (lane 2), 49G (lane 3), and 12E3 (lane 5) detected two bands located at approximately 46-48 and 84-86 kDa, while they were not detectable when 1251-labeled GEJ cell membranes were similarly treated with an unrelated mAb (lane 1) or when

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control cells were used instead of GEJ cells (data not shown). Moreover, when the same material was used for immunoprecipitation with hTSH-coupled CNBr-Sepharose beads, only the 46-kDa band was detected (data not shown). However, mAb llE7 (lane 4) behaved differently and detected one band located at 84-87 kDa; the 46- to 48-kDa band was very weak. Furthermore, when the same experiment was conducted under reducing conditions, identical profiles were observed for each of the mAb tested (data not shown). CAMP production (Fig. 8) and iodide accumulation (Fig. 9) in response to the four mAb were measured on collagenase-dissociated murine thyroid cells and compared to those obtained with one unrelated mAb and hTSH. We previously verified that mAb to hTSH-R bind similarly to murine and human thyroid cells (data not shown). In both assays, 1 mu/ml hTSH induced 41.3% stimulation of CAMP and 52% stimulation of iodide accumulation. Of the four mAb to hTSH-R, mAb 34A






control mAb


FIG. 8. Stimulation of CAMP production of murine thyroid cells by mAb to hTSH-R (100 pg/ml). Shown are the mean k SEM of three experiments.




i mAb


induced the highest functional activity; however, optimal iodide uptake was obtained with 0.5 pg/ml, whereas CAMP accumulation was reached with 100 pg/ml. In these experiments, mAb llE7 and 49G behaved quite differently. They induced 70-80% of the basal level of CAMP production by thyroid cells, but did not induce iodide accumulation. Lastly, one mAb to hTSH-R, 12E3, was totally inefficient in these two functional studies and behaved similarly to control mAb. Discussion

Functional studies of mAb to hTSH-R







FIG. 9. Stimulation of iodide accumulation of murine thyroid epithelial cells by mAb to hTSH-R. Shown are the mean + SEM of three experiments. n , 34A; 0,49G; 0, llE7; 0, 12E3; A, control mAb.

In the present study four mAbs to TSH-binding sites from GEJ cells (12) or human thyroid cells were selected from spleen cells of one DBA/l mouse previously extensively immunized with affinity-purified solubilized GEJ cell membranes. For that purpose, a first simple screening was performed using an ELISA toward the characterized GEJ cell TSH-binding site (10). The selected mAb were further tested for their binding capacities to intact GEJ cells or freshly dissociated human thyroid cells, using flow cytometry analysis. With each of the mAb, dose-effect responses were observed in terms of binding to purified hTSH-R as well as to intact cells. However, one mAb, 12E3, exhibited better binding to intact cells, suggesting that mAb 12E3 has an antibodybinding site that depends on the native conformation of the hTSH-R. Their binding to hTSH and other thyroid antigens, thyroid peroxidase and thyroglobulin, was negative. Their specificity for the TSH-binding site of thyroid cells was investigated. Solubilized membranes from porcine thyroid cells or stably transfected CHO cells JPO9 (13), which express only high levels of the recombinant hTSH-R, were also used to determine whether the mAb could interfere with the binding of [lz51]TSH to these membranes. Inhibition of binding of [lz51]bovine TSH to porcine membrane was dose dependent with only mAb 34A. In contrast, no significant inhibition was detectable when the three other mAb to hTSH-R were similarly used. Inhibition of binding of [‘251]hTSH to CHO cell membranes by mAb 12E3,11E7, and 49G was 52%, 65%, and 68%, respectively, suggesting that these mAb recognized an epitope of the hTSH-R located near or very near the hormone-binding site, whereas it was 99.6% for mAb 34A, demonstrating that it probably recognizes the hormone-binding site. These results suggest that TSHbinding sites would be very similar on porcine and human TSH-R, whereas modifications seem to have occurred in the near environment of the TSH-binding site of the two species. In a second set of experiments, mAb were covalently bound to CNBr-Sepharose to determine and compare the Mr characteristics of aliquot material from solubi-

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lized GEJ or human thyroid cell membranes bound to either various mAb or hTSH beads forming the columns. As predicted by our previous biochemical studies, major bands located at approximately 48 kDa were observed equally when using mAb or hTSH as ligands. However, for one of them, 34A, a weak 86-kDa protein band was also detected, corresponding to the minor band around 90 kDa that we previously described using hTSH as ligand (10). This band was also detectable when human thyroid cells were used instead of GEJ cells. In that respect, the very recent experiments (18) in which mAb to the LH receptor (LH-R) were used to purify LH-R from porcine testicular membranes must be considered. The researchers observed two LH-binding bands: a major one at approximately 85 kDa, and a minor one at approximately 45-50 kDa. We previously demonstrated, using mAb to LH-R (19), that LH-R and TSH-R share a common epitope(s), and we hypothesized the existence of sequence homologies at the protein and gene levels, which have now been confirmed (3-6, 20). These biochemical similarities between LH-R and TSH-R are not surprising, and they were further confirmed when mAb were used in Western blot or immunoprecipitation experiments with solubilized electrophoresed GEJ or human thyroid cell membrane proteins. Again, both strong 46- to 50-kDa and weak 86- to 88-kDa bands were regularly detected with three of the mAb to hTSH-R; mAb llE7 mainly immunoprecipitated proteins with a Mr of 86-88 kDa. This pattern of immunoprecipitation was obtained with solubilized GEJ cells under reducing as well as nonreducing conditions, excluding the existence of a disulfide bridge in hTSH-R from GEJ cells. Lastly, the induction of thyroid functions, such as iodide accumulation or CAMP production, after stimulation by mAb to hTSH-R was also tested. A relationship was established between [lz51]TSH binding to hTSH-Rtransfected CHO cells and the ability of mAb to induce thyroid function. 34A mAb, which fully competes with [1251]TSH for binding to human or porcine TSH-R, induces both thyroid functions. 12E3 mAb, which binds exclusively to hTSH-R and was the least potent inhibitor of [ lz51]TSH binding, never induces functional activities of thyroid cells. 49G and llE7, which inhibit approximately 65% of [‘251]TSH binding to hTSH-R-transfected CHO cells, can induce CAMP production only, never the thyroid-specific function of iodide accumulation. These data must be considered taking into account the recent clonings of TSH-R (3-6), which demonstrate a minimum 85 kDa Mr and the existence of two species of messenger coding for hLH-R (5) and hTSH-R (21), one containing the totality of the receptor (78-80 kDa) and the other containing only the extracellular domain (4850 kDa). In that respect, our original hypothesis (10) that hTSH-R expressed by GEJ cells are for the most


Endo. 1992 Vol 130. No 2


part truncated forms of the native hTSH-R is reinforced. The majority of GEJ cells seems to contain the mRNA encoding the extracellular domain of the hTSH-R. Truncation of GEJ cell hTSH-R could be either constitutive, and therefore preexisting in the patient thyroid cells before immortalization, or an artefact generated either during the hybridization or as the result of proteolysis during the solubilization and purification procedures. Compared to past studies in which mAb to hTSH-R have been selected from peripheral blood lymphocytes from Graves’ disease patients (22-24) and their selections made using either binding or functional studies on human thyroid cells or membranes, the specificity of these mAb for hTSH-binding sites was clearly established. In particular, we have been able to demonstrate the inhibition of binding of [‘251]TSH to CHO cells, which express only one thyroid-specific protein, the TSH-R. It is true that glycosylation of this CHO/TSHR may be different from that in thyroid cells, but that does not seem to interfere with the bindingpioactivity of TSH, TSAb, or TBAb (25). In conclusion, it can be assumed that these four mAbs to the hTSH-R presently described represent unique material, whose biological activities have to be tested to define the role of autoantibodies to hTSH-R in the induction of hyperthyroidism. Acknowledgments The authors thank Dr. Albert Louvel (Cochin Hospital) for providing human thyroid tissues. They also thank Mrs. Jocelyne Decaix for her excellent secretarial work. References 1. Burman KD, Baker Jr JR 1985 Immune mechanisms in Graves’ disease. Endocr Rev 6:183-232 2. Mariotti S, Chiovato L, Vitti P, Marcocci C, Fermi GF, Del Prete GF, Tiri A, Romagnani S, Ricci M, Pinchera A 1989 Recent advances in the understanding of humoral and cellular mechanisms implicated in thyroid autoimmune disorders. Clin Immunol Immunopathol5OS73S84 3. Parmentier M, Libert F, Maenhaut C, Lefort A, Gerard C, Perret J, van Sande J, Dumont JE, Vassart G 1989 Molecular cloning of the thyrotropin receptor. Science 246:1620-1622 4. Libert F, Lefort A, Gerard C, Parmentier M, Perret J, Ludgate M, Dumont JE, Vassart G 1989 Cloning, sequencing and expression of the human thyrotropin (TSH) receptor: evidence for binding of autoantibodies. Biochem Biophvs Res Commun 165:1250-1255 5. Misrahi M, Loosfelt H, Atger M, Sar S, Guiochon-Mantel A, Milgrom E 1990 Cloning, sequencing and expression of human TSH receptor. Biochem Biophvs Res Commun 166394-403 6. Nagayama Y, Kaufman KD; Seto P, Rapoport B 1989 Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor. Biochem Biophys Res Commun 165:1184-1190 I. Ohmori M, Endo T, Onaya T 1991 Development of chicken antibodies towards the human thyrotropin receptor peptides and their bioactivities. Biochem Biophys Res Commun 174:399-400 8. Endo T, Ohmori M, Ikeda M, Onaya T 1991 Thyroid stimulating activity of rabbit antibodies towards the human thyrotropin receptor peptide. Biochem Biophys Res Commun 177:145-150

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9. Libert F, Ludgate M, Dinsart C,, Vassart G, Thyroperoxidase but not the thyrotropin receptor contains sequential epitopes recognized by autoantibodies in recombinant peptides expressed in the pUEX vector. J Clin Endocrinol Metab, 73:857-86010. Remv JJ. Salamero J. Charreire J 1987 Covalent cross-linking of thyrotropin to its receptor on cloned hybrid human thyroid cells (GEJ). Endocrinology 121:1733-1741 11. Knudsen KA 1985 Proteins transferred to nitrocellulose for use as immunogens. Anal Biochem 147:285-288 12. Karsenty G, Michel-Bechet M, Charreire J 1985 Monoclonal human thyroid cell line GEJ expressing human thyrotropin receptors. Proc Nat1 Acad Sci USA 82:2120-2124 13. Perret J, Ludgate M, Libert F, Gerard C, Dumont J, Vassart G, Parmentier M 1990 Stable expression of the human receptor in CHO cells and characterization of differentially expressing clones. Biochem Biophys Res Commun 171:1044-1059 14. Bradford MM 1976 A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254 15. Kohler G, Milstein C 1975 Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497 16. Moxham CP, George ST, Graziano MP, Brandwein HJ, Malbon CC 1986 Mamalian beta-l and beta-2 adrenergic receptors. J Biol Chem 261:14562-14570 17. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 MT, Jolivet A, Jallal B, Salesse R, Bidart JM, 18. Vuhai-Luuthi Houllier A, Guiochon-Mantel A, Garnier J, Milgrom E 1990 Monoclonal antibodies against luteinizing hormone receptor. Immunochemical characterization of the receptor. Endocrinology 127:2090-




2098 F, Jallal B, Salesse R, Bidart JM, Remy JJ 19. Bedin C, Antonicelli 1989 Lutropin receptor and thyrotropin receptor share a common epitope. Mol Cell Endocrinol 65:135-144 20. McFarland K, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH 1989 Lutropin-choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science 245:494-499 21. Akamizu T, Ikuyama S, Saji M, Kosugi S, Kozak C, McBride OW, Kohn LD 1990 Cloning, chromosomal assignment, and regulation of the rat thyrotropin receptor: expression of the gene is regulated by thyrotropin, agents that increase CAMP levels, and thyroid autoantibodies. Proc Nat1 Acad Sci USA 87:5677-5681 22. Ealey PA, Valente WA, Ekins RP, Kohn LD, Marshall NJ 1985 Characterization of monoclonal antibodies raised against solubilized thyrotropin receptors in a cytochemical bioassay for thyroid stimulators. Endocrinology 116:124-131 23. Valente WA, Vitti P, Yavin Z, Yavin E, Rotella CM, Grollman EF, Toccafondi RS, Kohn LD 1982 Monoclonal antibodies to the thyrotropin receptor: stimulating and blocking antibodies derived from the lymphocytes of patients with Graves disease. Proc Nat1 Acad Sci USA 79:6680-6684 24. Yoshida T, Ichikawa Y, Ito K, Homma M 1988 Monoclonal antibodies to the thyrotropin receptor bind to a 56-kDa subunit of the thyrotropin receptor and show heterogeneous bioactivities. J Biol Chem 263:16341-16347 M, Perret J, Parmentier M, Gerard C, Libert F, Dumont 25. Ludgate J, Vassart G 1990 Use of the recombinant human thyrotropin receptor (TSH-R) expressed in mammalian cell lines to assay TSHR autoantibodies. Mol Cell Endocrinol 73:R13-R18

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Characterization of monoclonal antibodies to the human thyrotropin receptor.

We have produced four monoclonal antibodies (mAbs), 34A, 49G, 11E7, and 12E3, which bind the human TSH receptor (hTSH-R) when expressed on a human thy...
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