Estimation of Serum TSH Receptor AutoantibodyConcentration and Affinity

Nobuhiro Nakatake,1 Jane Sanders,1 Tonya Richards,1 Peter Burne,1 Carol Barrett,1 Chiara Dal Pra,2

Fabio Presotto,2 Corrado Betterle,3 Jadwiga Furmaniak,1 and Bernard Rees Smith1

We have used the human monoclonal TSH receptor (TSHR) autoantibody (M22) as a labeled ligand in com-petition with individual patient TSHR autoantibodies (TRAb) to estimate their serum concentrations and affi-nities. TSHR coated tubes, 125I-labeled M22 IgG and Fab, and patient sera IgG and Fab were used in thesestudies. In 15 patients with Graves’ disease, TRAb concentrations ranged from 50 to 500 ng=mL of serum (5–60 parts per million of total serum IgG) and TRAb IgG affinities from 3.0 ± 1.0� 6.7 ± 1.54�1010 L=mol (mean±SD; n¼ 3). Fab fragment affinities were similar to those of intact IgG. Serum TRAb with blocking (TSH an-tagonist; 4 patients) activity had similar affinities (3.0 ± 0.25� 7.2 ± 2.2�1010 L=mol) to TRAb IgG from patientswith Graves’ disease, but blocking TRAb concentrations were higher (1.7 – 27 mg=mL of serum). The con-centrations of TRAb that we observed in the sera of the 15 Graves’ patient (0.33 – 3.3 nmol=L) can be comparedwith that of circulating TSH. In particular, a serum TSH concentration of 100mU=L (0.7 nmol=L) is in the samerange as the concentrations of TRAb we observed. Such a TSH concentration (similar to that observed afterinjection of 0.9mg of recombinant human TSH) would be expected to cause a similar degree of thyrotoxicosis asseen in Graves’ disease. Consequently, the thyroid-stimulating potencies (i.e., activity per mol) of patient serumTRAb and human TSH appear to be of a similar magnitude in vivo as well as in vitro. Overall, our results indicatethat serum TRAb affinities are high and show only limited variations between different sera whereas con-centrations of the autoantibodies vary widely.

Introduction

Autoantibodies to the TSH receptor (TSHR) are presentin small amounts in patient sera and bind to the recep-

tor with high affinity (1). Previous methods used to assessthese two parameters have been based on affinity purifica-tion of serum TSHR autoantibodies using native (2,3) and re-combinant (4,5) TSHR preparations. These earlier approacheshave some limitations, and we now describe a new approach,namely, the human monoclonal TSHR autoantibody (M22)(6,7), which has beenused as a labeled ligand for binding to theTSHR in competition with patient sera TSHR autoantibodies.These studies have allowed the most detailed assessment ofserum TSHR autoantibody affinity and concentration to date.

Materials and Methods

Patient sera

Sera from 15 patients with Graves’ disease (with differentdisease duration and receiving different forms of treatment)

and four patients with hypothyroidism and high levels ofTSHR antagonist (blocking) autoantibodies were studied(Table 1). All sera were positive for TSHR autoantibodies asassessed by inhibition of M22 binding to the TSHR (8) (kitfrom RSR Ltd, Cardiff, UK). The TRAb levels ranged from1.5 to 1290 U=L (Table 1). In addition, all serum sampleswere tested for autoantibodies to thyroid peroxidase (TPO)and thyroglobulin (Tg) (Table 1) using immunoprecipitationassay kits from RSR Ltd (9). Sera from 10 healthy blooddonors (HBD; Golden West Biologicals, Vista, CA) with nodetectable TSHR autoantibodies were used as controls. In-formed consent for the study was obtained from the patients.

Analysis of thyroid-stimulating and -blocking activities

Stimulation of cyclic AMP production by patient sera andblocking of TSH stimulated cyclic AMP production by pa-tient sera were measured using CHO cells expressing thehTSHR (approximately 50,000 receptors per cell) (10). Briefly,CHO cells were seeded into 96-well plates (30,000 cells per

1FIRS Laboratories, RSR Ltd, Llanishen, Cardiff, United Kingdom.2Third Unit of Internal Medicine, Department of Medical and Surgical Sciences, University of Padua, Padua, Italy.3Unit of Endocrinology, Department of Medical and Surgical Sciences, University of Padua, Padua, Italy.

THYROIDVolume 16, Number 11, 2006# Mary Ann Liebert, Inc

1077

well) and incubated for 48 h in Dulbecco’s Modified Eagle’sMedium (DMEM; Invitrogen, Paisley, UK) containing 10%fetal calf serum. The DMEM was then removed and testsamples were added and incubated for 1 h at 378C. 100mL ofa 1 in 10 dilution of patient serum was assayed for stimu-lation of cyclic AMP production while 50mL of porcine TSH(RSR) and 50mL of diluted patient serum were used in theblocking assay (final concentration of TSH¼ 0.5 ng=mL; finalserum dilution¼ 1 in 10). All samples were diluted in NaCl-free Hank’s buffered salt solution containing 1 g=L glucose,20mmol=L HEPES, 222mmol=L sucrose, 15 g=L bovine se-rum albumin (BSA), and 0.5mmol=L 3 isobutyl-1-methyl-xanthine (pH 7.4; cyclic AMP buffer).

After 1 h of incubation and removal of the test material,cells were lysed and intracellular cyclic AMP levels mea-sured using a Biotrak enzyme immunoassay system (GEHealthcare, Chalfont St Giles, UK). For stimulation of cyclicAMP production by patient sera, results were expressed as apercentage of cyclic AMP levels observed in the presence of a1 in 10 dilution of a pool of HBD sera (>180% is positive). Forblocking the stimulation of cyclic AMP production, resultswere expressed as percentage inhibition of TSH stimulationin the presence of a HBD pool (1 in 10 final dilution) (in-hibition of >30% is positive).

Preparation of IgG and Fab fragments

IgGs were isolated from patient serum using affinitychromatography on MAbselect (GE Healthcare, Chalfont StGiles, UK), according to the manufacturer’s instructions, andstored in aliquots at �708C. All IgG preparations used in thestudy were greater than 95% pure, as assessed by analysis onSDS-PAGE (11).

IgG was treated with immobilized papain (0.2 U enzymeto 1mg IgG; Sigma Aldrich, Poole, UK) at 338C for 4 h. Thedigest was then passed through a MAbselect column to re-move any undigested IgG or Fc fragment from the Fab

preparation (12) and dialysed against phosphate-bufferedsaline (PBS; 137mmol=L NaCl, 8.1mmol=L Na2HPO4,2.7mmol=L KCl, 1.47mmol=L KH2PO4, 3.1mmol=L NaN3,pH 7.4). Purified Fab preparations (stored in aliquots at�708C) were greater than 95% pure by SDS-PAGE (11).

M22 IgG was obtained from heterohybridoma culturesupernatants, M22 Fab prepared as described previously (7)and stored in aliquots at �708C. Both M22 IgG and M22 Fabwere greater than 95% pure as judged by analysis on SDS-PAGE (11). Analysis on isoelectrofocusing gels (Bio-RadHemel Hempstead, UK) in the range of pH 3.0 to 10.0, ac-cording to the manufacturer’s instructions, indicated thatM22 IgG had a pI range of approximately 7.6–8.2 and M22Fab a pI range of approximately 8.0–8.2. M22 IgG and Fabwere labeled with 125I as described previously (7).

The concentration of preparations of M22 IgG or IgGpurified from different patient sera was determined by ab-sorbance at 280 nm on the basis that 1 mg of IgG or Fab permL¼ 1.40 absorbance units. The concentration of IgG inserum samples was measured by Boehring nephelometry(normal IgG concentration range is 5.1–15.8 g=L).

Estimation of the amount of TRAb inpurified patient IgGs

The amount of patient serum TRAb per milligram of pa-tient IgG was estimated using a calibration curve preparedfrom M22 IgG (2–500 ng=mL). Briefly, 50mL of diluted pa-tient IgG (5–5000 mg=mL) or M22 IgG were incubated inTSHR coated tubes (RSR Ltd) with 50 mL of 125I-labeled M22IgG (50,000 cpm) at room temperature for 120min with shak-ing. The tubes were then aspirated, washed twice with 1mLof coated tube (CT) assay buffer (50 mM NaCl, 10 mM Tris-HCl pH 7.8, 0.5% Triton X-100) and counted in a gammacounter. Inhibition of labeled M22 binding was calculated as100� (1 – (cpm bound in the presence of the test sample)=(cpm bound in the presence of CT assay buffer only)). The

Table 1. Characteristics of TRAb Positive Patients

Patient number Diagnosis Serum TRAb level (U/L) Serum TPO Ab (U/mL) Serum Tg Ab (U/mL) Medication

G1 Graves’ 110 8.5 6.0 L-thyroxineG2 Graves’ 50 307 neg MethimazoleG3 Graves’ 39 >500 135 MethimazoleG4 Graves’ 62 >500 neg MethimazoleG5 Graves’ 28 >500 neg MethimazoleG6 Graves’ 25 >500 675 naG7 Graves’ 29 >500 neg MethimazoleG8 Graves’ 26 19 neg NoneG9 Graves’ 25 234 neg MethimazoleG10 Graves’ 14 1.5 neg MethimazoleG11 Graves’ 20 91 746 MethimazoleG12 Graves’ 10 >500 109 MethimazoleG13 Graves’ 7.4 4.0 neg MethimazoleG14 Graves’ 5.8 >500 49 MethimazoleG15 Graves’ 1.5 15 8.0 L-thyroxine

H16 Hypothyroidism 953 42 81 L-thyroxineH17 Hypothyroidism 1290 >500 675 L-thyroxineH18 Hypothyroidism 188 245 168 naH19 Hypothyroidism 134 neg 779 L-thyroxine

TRAb levels measured by inhibition of M22 binding (8) are shown as U=L of reference preparation 90=672 from the National Institute forBiological Standards and Control (NIBSC); TPO Ab and Tg Ab levels are shown as U=mL of NIBSC reference preparations 66=387 and65=093, respectively. na, information not available; neg, undetectable.

1078 NAKATAKE ET AL.

amount of TRAbFab present in Fab purified frompatient IgGswas estimated using the same assay procedure, except thatunlabeled and 125I-labeled M22 Fab preparations were used.

The percent inhibition of labeled M22 binding causedby unlabeled M22 was plotted against M22 concentration(ng=mL) to give a calibration curve of the type shown inFigure 1. Similarly, percent inhibition of labeled M22 bindingcaused by patient serum IgG was plotted against patientserum IgG concentration (ng=mL), also shown in Figure 1.The concentrations of M22 that caused the same inhibitionsas patient serum IgG were then read off at three differentpoints on the M22 calibration curve and the value expressedas a mean. Thus, the TRAb levels in patient serum IgG areexpressed as their equivalent concentration in terms of M22concentration. Interassay precision studies (n¼ 6) for thisprocedure gave coefficients of variation of 7.6% at 11 ng=mL,5.9% at 17 ng=mL, and 3.6% at 56 ng=mL. Mean (� SD) in-hibitions of labeled M22 binding for these determinationswere 24.5� 2.43%, 34.7� 2.07, and 68.5� 0.55, respectively.

Association and dissociation studies

The association and dissociation of M22 IgG, M22 Fab,and TSH binding to the TSHR were carried out using TSHRcoated to plastic tubes (RSR Ltd). Highly purified porcineTSH (80U=mg) was used in these studies either labeled with125I or unlabeled (both preparations fromRSR). Tubes used forlabeled TSH binding were coated with 20� and 10� higherconcentrations of TSHR than tubes used for labeled M22IgG binding or labeled M22 Fab binding, respectively. In as-sociation experiments, 100 mL of 125I-labeled M22 IgG, 100mLof 125I-M22 Fab, or 100 mL of 125I-TSH were incubated in theTSHR coated tubes at room temperature with shaking for5–180min. The tubes were then aspirated, washed with2�1mL of CT assay buffer, and counted. For the dissociationexperiments, 100 mL of 125I-M22 IgG, 100mL of 125I-M22 Fab,or 100mL of 125I-TSH were incubated in the TSHR coatedtubes at room temperature for 180minwith shaking, followedby the addition of 10 mL of 1mg=mL M22 IgG or 1mg=mLM22 Fab, or 100mU=mL of TSH. After 0–180min of furtherincubation at room temperature with shaking, the tubes wereaspirated, washed as above, and counted.

A second method to study the dissociation of 125I-TSHfrom TSHR coated tubes by TSH, M22 Fab, M22 IgG, and

TSHRs was also used. Association of 100 mL 125I-TSH wascarried out as above for 180min, followed by aspiration ofthe 125I-TSH tracer and addition of 1mL CT assay buffercontaining 10 mL of 1mg=mL M22 IgG or 1mg=mL M22 Fabor 100mU=mL of TSH or detergent solubilized TSHR or CTassay buffer. After 0–180min of further incubation at roomtemperature with shaking, the tubes were aspirated, washedas above, and counted. The nonspecific binding for theseexperiments was taken as the binding of labeled TSH, M22IgG, or M22 Fab to tubes not coated with receptor. Thebinding was low (2–2.6% of total cpm added) and was notsubtracted from the results presented.

Scatchard analysis

Purified patient IgGs and Fab fragments were used toinhibit binding of 125I-M22 IgG or Fab, respectively, to theTSHR and their affinities estimated by Scatchard analysis(13). Briefly, 50mL aliquots of unlabeled, purified patient IgGor Fab (containing 0.5–500 ng=mL TRAb, estimated as de-scribed above) in CT assay buffer were incubated in TSHRcoated tubes (RSR Ltd) in duplicate with 50 mL of 125I -M22IgG or 125I-Fab (50,000 cpm) at room temperature with shak-ing. After 120min, the tubes were aspirated, washed twicewith 1mL of CT assay buffer, and counted in a gammacounter. A plot of bound against bound-free was used toestimate the affinities of the patients’ IgG and Fab fragments.

Results

Concentration of TRAb in patient sera

The amount of patient serum TRAb per mg of patientIgG (read off a calibration curve prepared from M22 IgG,as shown in Fig. 1) ranged from 5 to 2000 ng=mg in thedifferent sera studied (Table 2). The concentration of totalIgG in the patient sera ranged from 7.9 to 16.1 g=L, and theseconcentration values were used to calculate the amount ofTRAb per mL of patient serum. The levels ranged from 50 to500 ng=mL for the 15 Graves’ sera, and 1.7 to 27 mg=mL forthe four blocking sera (Table 2).

In the case of the 15 Graves’ sera, the actual concentrationof the TRAb in ng=mL in each serum sample correlated withthe serum U=L values obtained by inhibition of M22 binding(r¼ 0.90; p<0.001) (Fig. 2A) and with the TSHR stimulatingactivity of the sera (r¼ 0.88; p<0.001) (Fig. 2B).

Association and dissociation of TSH, M22 IgG,and M22 Fab binding to the TSHR

Binding of 125I-TSH to TSHR coated tubes was studiedover a 180min time period. As shown in Figure 3A, 125I-TSHbinding reached a plateau (about 28% of total cpm bound) atabout 90min and remained essentially unchanged for theremaining observation period. However, bound 125I-TSHdissociated quickly after the addition of 100mU=mL of un-labeled TSH, 1mg=mL of M22, or 1mg=mL of M22 Fab (Fig.3B). The addition of M22 IgG or Fab (1mg=mL) resulted in asimilar reduction in 125I-TSH binding to those observed afteraddition of unlabeled TSH (100mU=mL) (Fig. 3B).

Labelled M22 Fab binding to TSHR coated tubes reached aplateau at about 2 h (Fig. 3A) whereas 125I-M22 IgG bindingtook slightly longer to reach a plateau. Extending incubation

1

M22 IgG

G8 IgG

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Concentration of unlabelled IgG (ng/mL)

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FIG. 1. Estimates of amount of TRAb in purified IgG pre-parations (patients G1 and G8) using M22 IgG in a calibra-tion curve. ¼M22 IgG; ¼G1 IgG; ¼G8 IgG.

TRAB CONCENTRATION AND AFFINITY 1079

times with IgG or Fab beyond 3h did not result in furtherincreases in binding to the TSHR coated tubes (data notshown).

The addition of unlabeled M22 IgG (1mg=mL) or TSH(100mU=mL) to 125I-M22 IgG bound to TSHR coated tubesdid not cause effective dissociation even after 180min ofincubation (Fig. 3C). However, incubation with unlabeledM22 Fab (1mg=mL) caused some dissociation of 125I-M22IgG from the TSHR coated tubes (Fig. 3C).

Binding of 125I-M22 Fab to TSHR coated tubes was alsoessentially unaffected by incubation with 1mg=mL un-labeled M22 IgG or TSH (100mU=mL) (Fig. 3D). However,as with labeled M22 IgG, some dissociation of 125I-M22 Fabbinding was observed following incubation with unlabeledM22 Fab (Fig. 3D).

Dissociation of 125I-labeled TSH bound to TSHR coatedtubes (bound during a 3 h incubation) was also studied afterwashing the tubes and the addition of various materials toinhibit reassociation of dissociated labeled TSH. As can beseen in Figure 4, the extent and rate of dissociation wereincreased relative to buffer only in the presence of M22 IgGand Fab (greatest effect) and in the presence of TSH and de-tergent solubilized TSHR (weakest effect). For example, after180min, about 38% of the original TSH bound dissociated inthe presence of buffer only. This increased to 63, 74, 80, and82% in the presence of TSHR, unlabeled TSH, M22 IgG, andM22 Fab, respectively (Fig. 4).

Affinity of TRAbs for the TSHR in different patient sera

Representative Scatchard plots for purified IgG and Fabfrom patient G1 binding to the TSHR are shown in Figure 5.

The estimated affinity of G1 IgG, 3.0�1010 L=mol, wassimilar to the affinity of G1 Fab (1.7�1010 L=mol) (Fig. 5 andTable 2). Estimated affinity constants for TRAb IgGs fromthe sera of 15 Graves’ patients (G1–15) ranged from3� 0.05�1010 L=mol to 6.7� 1.54�1010 L=mol (Table 2). Fabpreparations obtained from a further four Graves’ patientsera IgGs (G2, G6, G7, and G8) showed affinities similar tothose calculated for their respective intact IgGs (Table 2). Theaffinity constants estimated for the four blocking TRAbs(3.3� 0.24�1010 L=mol, 3.0� 0.25�1010 L=mol, 3.2� 0.94�1010 L=mol, and 7.2� 2.2�1010 L=mol) were similar to theaffinity constants found for TRAbs in Graves’ sera (Table 2).Furthermore, the Fab preparations derived from the blockingTRAb IgGs also showed similar affinities to their respectiveintact IgGs (Table 2). The affinity of TSH for the TSHR coatedtubes was 3.5� 0.68�109 L=mol (n¼ 3).

Discussion

Analysis of the proportion of TRAb in the 15 differentGraves’ patient IgG preparations we analyzed indicated arange of 5–60 parts per million of the total serum IgG (5–60 ng=mg for G1–15 as shown in Table 2). This is somewhatlower than the proportion of TRAb in the serum IgG of theM22 lymphocyte donor (i.e., 300 parts per million). The 4sera IgGs with TSH antagonist activity (H16–19 in Table 2)had a higher proportion of TRAb (120–2000 parts per mil-lion) than the Graves’ sera IgGs. The higher concentration ofTRAb in these blocking sera presumably reflects a strongerimmune response to the TSHR. Why this tends to lead toautoantibodies that act as antagonists rather than agonists isnot clear at present.

Table 2. Estimates of TRAb Concentration and Affinity for TSHR in 18 Patient Sera

Patient

Proportion of TRAbin purified patient

IgG (ng/mg)

TRAb concentrationin patient serum

(ng=mL)

TRAb IgG affinity(�1010 L/mol)

(mean� SD; n¼ 3)

TRAb Fab affinity(�1010 L/mol)

(mean� SD; n¼ 3)Stimulatingactivity (%)a

G1 60 480 3.3� 0.30 2.0� 1.20 2162G2 17 210 4.0� 0.80 2.9� 0.85 257G3 12 190 4.9� 0.20 nd 98G4 35 500 4.1� 0.10 nd 1867G5 14 140 5.8� 0.80 nd 233G6 21 160 6.7� 1.54 2.8� 0.37 1181G7 16 160 3.0� 1.00 1.7� 0.55 260G8 15 170 3.6� 0.80 2.7� 0.15 107G9 14 130 3.0� 0.05 nd 640G10 6 97 3.3� 0.90 nd 114G11 19 200 3.9� 0.40 nd 911G12 8 94 4.3� 0.95 nd 106G13 7 87 4.6� 0.75 nd 179G14 9 69 3.8� 0.10 nd 143G15 5 50 3.2� 0.35 nd 52

Blocking activity (%)b

H16 2000 27000 3.3� 0.24 4.5� 2.33 96H17 410 6600 3.0� 0.25 2.0� 0.53 57H18 320 4400 3.2� 0.94 2.7� 0.90 91H19 120 1700 7.2� 2.2 2.3� 0.12 79

aStimulation of cyclic AMP production in TSHR expressing CHO cells by 1 in 10 dilutions of patient sera; values >180% are positive.bInhibition of TSH stimulation of cyclic AMP production in TSHR expressing CHO cells by 1 in 10 dilutions of patient sera; values >30%

are positive. Sera H17 and H19 showed stimulating activity when assayed at a 1:10 dilution without TSH (i.e., 678 and 530%, respectively).M22 IgG affinity: 4.5� 1.18�1010 L/mol (mean� SD; n¼ 15). M22 Fab affinity: 3.4�1.43�1010 L/mol (mean� SD; n¼ 7); nd, not

determined. M22 lymphocyte donor IgG contained 300ng TRAb per mg of IgG (for clinical details, see Sanders et al. [6]).

1080 NAKATAKE ET AL.

A study of the association of labeled TSH, M22 IgG, andM22 Fab to TSHR coated tubes (Fig. 3A) indicated that theTSH bound more rapidly than M22 Fab, which bound morerapidly than M22 IgG (half maximal binding occurred after17, 32, and 52min, respectively). The addition of unlabeledTSH,M22 IgG, orM22 Fab to the incubationmixtures resultedin dissociation of bound labeled TSH, with 50% of the boundhormone dissociating in approximately 20min. Detectabledissociation of labeled M22 IgG or Fab did not occur whenunlabeled TSH or M22 IgG were added, but the addition ofunlabeledM22 Fab resulted in dissociation of labeledM22 IgGand labeled M22 Fab, with 50% of the bound material dis-sociating in approximately 3 h. This relative slow dissociationrate of M22 from the TSHR is characteristic of antibody-anti-gen interactions (15). Although the reason for this difference inthe effects ofM22Fab and IgG is not clear at present, it could berelated to the faster rate of the Fab.More rapid binding ofM22Fab to the TSHR compared toM22 IgG could be due in part atleast to the higher isoelectric point of the Fab, which might beexpected to enhance the charge-charge interactions, which areimportant in the binding of the TSHR to both TSH and TSHR

autoantibodies (1). These features of the M22 Fab-TSHR in-teraction also provide a possible explanation as to why M22Fab is a more potent thyroid stimulator than is M22 IgG (7).

Although there was a clear overall correlation betweenserum TRAb concentration in ng=mL and the ability of eachserum to stimulate cyclic AMP production in TSHR trans-fected CHO cells, there were some apparent discrepancies. Inparticular, in some cases, sera with similar levels of TRAbshowed markedly different levels of stimulating activity. Thereason for this is not clear at present but it could reflect dif-ferences in the ability of individual TSHR autoantibodies toinitiate a stimulating signal once they have bound to theTSHR. This would be analogous to the now well-establisheddifferent stimulating activities of different monoclonal anti-bodies to the TSHR (6,7,16–18). Alternatively, or in addition,the patient sera could contain a mixture of stimulating andblocking autoantibodies with samples showing stronger stim-ulating activity for the same TRAb concentration containinga lower proportion of blocking autoantibodies. It is also pos-sible that inhibition of M22 binding assays and=or inhibitionof TSH binding assays do not detect some TSHR auto-antibodies, which bind to the TSHR and cause stimulation.However, there is as yet no good evidence in the literaturefor the existence of patient TRAb with these characteristics.

A recent study (14) has concluded that the interaction be-tween labeled TSH and TSH receptors on the surfaces of in-tact CHO cells is characterized by negative co-operativity,that is, the addition of unlabeled TSH (or an unlabeled mousemonoclonal thyroid-stimulating antibody) actively causeddissociation of bound TSH in addition to passively blockinglabeled TSH binding. Such a phenomenon does not appear tobe occurring in the TSHR coated tube system we have used,as the addition of detergent solubilized TSH receptors to la-beled TSH bound to the coated tubes promoted dissociationin a similar way to that of unlabeled TSH (Fig. 4).

The affinities for the TSHR of all 19 patient IgGs studiedwere similar as judged by Scatchard analysis and were about10 times higher than the affinity of TSH. Furthermore, in the9 cases where IgG and Fab affinity were compared, the di-valent and monovalent autoantibody preparations had sim-ilar affinities, with the average ratio of Fab=IgG affinity being0.7. This suggests that the intact IgG preparations, althoughdivalent, tend to interact monovalently with the TSHR (15),at least in the coated tube system we used. Also, the affinitiesof M22 IgG and M22 Fab were well within the range of af-finities observed for the various patient serum preparations,indicating that M22 is quite representative of patient serumTSHR autoantibodies.

The results we have obtained on the affinities and con-centrations of TSHR autoantibodies using M22 as a re-presentative ligand can be compared with other studies. Forexample, reports from the 1970s (2) and 1980s (3) using af-finity purification indicated that TSHR autoantibodies were avery small proportion of the total patient serum IgG, andconsideration of the results obtained at that time (2,3) sug-gests that in the region of 1 part per 1000 of particularlypotent sera IgGs was TSHR autoantibodies. This is a similarorder of magnitude to the values we have obtained for serawith very high TSHR autoantibody levels (Table 2). Morerecently, these earlier studies have been extended by usingrecombinant TSH receptor preparations to affinity purifyTSHR autoantibodies (4,5). However, purification still

y = 0.1734x+ 86.408r = 0.88n = 15p <0.001

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FIG. 2. Relationship between serum TRAb concentration inng=mL in 15 Graves’ sera and (A) TRAb levels in U=L ofNIBSC 90=672 measured by inhibition of M22 binding. (B)Stimulation of cyclic AMP production (%) in TSHR trans-fected CHO cells by each serum. Inclusion of the 4 blockingsera in the correlation shown in A gives y¼ 13.217xþ 134.31;r¼ 0.74; p< 0.001. Correlation of TRAb U=L vs percentstimulation for the 15 Graves’ sera gives an r value of 0.78( p< 0.001).

TRAB CONCENTRATION AND AFFINITY 1081

appeared to be incomplete and some denaturation of the au-toantibodies may have occurred during the relatively harshconditions used to elute receptor bound antibody. This couldexplain why the affinities reported for TSHR autoantibodies(in the region of 109 L=mol) were lower than those we ob-served, and the proportion of TRAb in purified patient IgG

was somewhat higher than we observed. In another study(19), isolated recombinant TSHR A subunit was used to ti-trate the levels of TRAb in patient sera. This gave estimatesof the TRAb concentrations in the range 0.1–1mg=mL, con-sistent with the results we report here. Also, Atger and col-leagues (20), using a conventional sandwich ELISA based onplate wells coated with affinity-purified recombinant TSHRand peroxidase-labeled anti-human IgG (or anti-humanIgM), reported an affinity of 1–4�1010 L=mol (and con-centrations in the region of 3.5 mg=mL) for TRAb in Graves’patient sera. However, Atger et al. (20) found that 55% ofhealthy blood donor sera contained TRAb with similar affin-ity to the TRAb in Graves’ sera (also at high concentration,i.e., in the region of 1.8 mg=mL); consequently it is difficult tointerpret these findings.

The procedures we describe here are simple, require re-latively small volumes of sera, and can be performed quicklyon a number of different serum samples. This contrastsmarkedly with the extensive experimentation required foraffinity purification of patient sera or titration studies withisolated TSHR A subunit. The convenience of our proceduremakes it suitable for studying changes in TRAb affinity andconcentration during the course and treatment of Graves’disease.

The on rate (k1) for M22 Fab binding to the TSHR oncoated tubes was estimated from the initial reaction rate (Fig.3A) (15) to be 1.2�1010 M-1 hr-1 and the off rate (k-1) for M22Fab dissociation from the TSHR bound to coated tubes (Fig.3D) (15) was 4.2�10-2 hr-1. These results gave an associationconstant (K) value of 2.9�1011 L=mol for M22 Fab, which is

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A B

C D

FIG. 3. Kinetics of 125I-labeled TSH, M22 IgG, and M22 Fab interacting with TSHR coated tubes (see text for details). (A)Time course of binding of 125I-labeled TSH, M22 IgG, and M22 Fab (nonspecific binding, i.e., binding of labeled TSH, M22IgG, or M22 Fab to tubes not coated with the TSHR was low (2–2.6% of total cpm added) and was not subtracted from thedata shown in the figure). (B) Dissociation of labeled TSH binding. (C) Dissociation of labeled M22 IgG binding. (D)Dissociation of M22 Fab binding in the presence of unlabeled TSH (), M22 IgG (), M22 Fab () or CT assay buffer only ().Unlabeled materials (10mL) were added to the coated tubes containing 100 mL of labeled ligands after an initial incubation of3 h at room temperature. The cpm bound were then followed for a further 3 h at room temperature.

0

5

10

15

20

25

30

12

5I-

TS

H b

ou

nd

(%

of

tota

l cp

m)

0 30 60 90

Incubation time (minutes)

120 150 180

FIG. 4. Dissociation of 125I-TSH from TSHR coated tubes inthe presence of M22 IgG (), M22 Fab (), TSH (), detergentsolubilized TSHR (), and CT assay buffer (). TSHR coatedtubes were incubated with 125I-labeled TSH for 3 h, the traceraspirated, CT assay buffer containing TSHR ligands or de-tergent solubilized TSHR added, and the amount of labeledTSH remaining bound followed over 180min (see text forexperimental details).

1082 NAKATAKE ET AL.

8 times higher than the value of 3.4�1010 L=mol obtained viaScatchard analysis (Table 2). The reason for this discrepancyis not clear at present but may well reflect the difficulty inassessing the initial reaction rate (i.e., the slope of the asso-ciation curve in Fig. 3A) accurately.

The concentration of TSHR autoantibodies of 50–500 ng=mL or 0.33–3.3 nmol=L that we observed in the 15 Graves’patient sera can be compared with that of circulating TSH.100mU=L of TSH is approximately 20 ng=mL or 0.7 nmol=L(i.e., the concentrations of TSHR autoantibodies that we ob-served in the 15 Graves’ sera are in the same range as100mU=L of TSH). Such a TSH concentration (similar to thatobserved after injection of 0.9mg of recombinant humanTSH (21–25) would be expected to cause a similar degree ofthyrotoxicosis as seen in Graves’ disease. Consequently, thethyroid-stimulating potencies (i.e., activity per mol) of pa-tient serum TRAb and human TSH appear to be of a similarmagnitude in vivo as well in vitro.

Overall our results provide detailed estimates of affin-ity and concentration of serum TSHR autoantibodies. Affin-ities are high and show very limited variation betweendifferent sera whereas concentrations of the autoantibodiesvary widely between different sera.

Acknowledgments

We are grateful to Carol James for her expert preparationof the manuscript. Dr. Nakatake is a recipient of an RSRFellowship. RSR Ltd is a developer of medical diagnostics,including kits for measuring thyroid autoantibodies.

Disclaimer

Terry F. Davies, MD, the Editor-in-Chief of Thyroid is aconsultant to RSR.

References

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Ka = 1.7 x 1010 L/molKa = 3.0 x 1010 L/mol

A B

Bo

un

d/F

ree

0

0.2

0.4

0.6

0.8

Bo

un

d/F

ree

0

0.2

0.4

0.6

0.8

0

Bound IgG (ng) Bound Fab (ng)

0.2 0.4 0.6 0 0.1 0.2 0.3

FIG. 5. Scatchard analysis of IgG (A) and Fab (B) from patient G1 using TSHR coated tubes and 125I-labeled M22 IgG orFab, respectively. The concentration of TRAb in the patient IgG and Fab were estimated using calibration curves of the typeshown in Figure 1. The amount of IgG (or Fab) bound was estimated by multiplying these concentrations by the proportionof labeled M22 (or Fab) bound (see text for experimental details).

TRAB CONCENTRATION AND AFFINITY 1083

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Address reprint requests to:Bernard Rees Smith

FIRS Laboratories

RSR Ltd

Parc Ty Glas

Llanishen

Cardiff, CF14 5DU

United Kingdom

E-mail: firs@rsrltd.eclipse.co.uk

1084 NAKATAKE ET AL.

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