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Demonstration of Immunoglobulin G, A, and E
Autoantibodies to the Human Thyrotropin Receptor
Using Flow Cytometry
RUSSELL METCALFE, NICOLA JORDAN, PHILIP WATSON, SEVIM GULLU, MARIE WILTSHIRE,
MICHELE CRISP, CAROL EVANS, ANTHONY WEETMAN, AND MARIAN LUDGATE
Department of Medicine, Clinical Sciences Center, Northern General Hospital (R.M., P.W., A.W.), Sheffield, United Kingdom
S5 7AU; and Departments of Medicine (N.J., S.G., M.C., M.L.) and Pathology (M.W.), University of Wales College of
Medicine, and Department of Medical Biochemistry, University Hospital of Wales National Health Service Trust (N.J.,
C.E.), Heath Park, Cardiff, United Kingdom CF14 4XN
Human TSH receptor (TSHR) autoantibodies with biological
activity result in thyroid dysfunction, but antibodies that sim-
ply bind do not. We have applied flow cytometry to the mea-
surements of IgG, IgA, and IgE immunoreactivity to the TSHR
in patients with Graves’ disease (GD) and thyroid eye disease
(TED) and in normal controls.
CHO cells stably expressing the extracellular domain of the
TSHR with a glycophosphatidylinositol anchor were pro-
duced and found to express approximately 4 times as many
receptors, but of similar affinity, as JP09 in TSH binding stud-
ies. Substantial increases in median fluorescence and peak
channel fluorescence were obtained by flow cytometry using
TSHR monoclonal antibodies on the glycophosphatidylinosi-
tol cells.
IgG autoantibodies were demonstrated in 55 of 65 un-
treated GD patients, 3 of 25 normal subjects, and 4 of 8 atypical
TED sera (negative for TSHR autoantibodies with biological
activity) by flow cytometry and correlated poorly with thy-
roid-stimulating antibodies. IgA antibodies were present in 1
of 12 normal, 1 of 7 treated GD with TED, and 3 of 8 atypical
TED sera. IgE binding was observed in 1 of 12 normal, 2 of 8
treated GD without TED, 1 of 6 treated GD with TED, and 0 of
8 atypical TED sera.
In conclusion, we have demonstrated autoantibodies that
bind directly to the TSHR in the majority of GD patients and
in 50% of patients with atypical TED and a small number of
normal controls lacking TSHR antibodies that affect function.
Although predominantly IgG
, TSHR autoantibodies of the
IgA and IgE isotypes are also detectable. (J Clin Endocrinol
Metab 87: 1754 –1761, 2002)
AUTOANTIBODIES to the TSH receptor (TSHR) are
found in patients with autoimmune thyroid disor-
ders, particularly Graves’ disease (GD). They are classified
according to their biological activity. Thyroid-stimulating
antibodies (TSAB) mimic the action of TSH and result in
hyperthyroidism. Thyroid-blocking antibodies (TBAB) in-
hibit TSH induced stimulation and may be the cause of
hypothyroidism in idiopathic myxedema. Both TSAB and
TBAB may also be TSH binding-inhibiting Igs (TBII), which
prevent hormone/receptor interaction (1).
Assays in current diagnostic use are not able to detect
antibodies that simply bind to the receptor without any effect
on the TSHR or TSH binding. The other major autoantigens
in autoimmune thyroid disease are Tg and thyroperoxidase
(TPO); both can be detected by direct binding, e.g. in ELISA
(2). The incidence of TPO antibodies would be grossly un-
derestimated if the only detection methods relied on a bio-
logical effect such as inhibition of enzyme activity. Direct
binding has also revealed that 10–20% of normal euthyroid
individuals, especially women, have circulating Tg and TPO
autoantibodies, but little has been reported regarding TSHR
antibodies in such a control population (3).
Previous studies investigating direct binding of autoanti-
bodies to the TSHR have concentrated on sera from known
TSAB- and/or TBII-positive GD patients. A variety of meth-
ods have been used, including ELISA, in vitro transcription/
translation, Western blotting of bacterially produced TSHR,
and flow cytometric analysis of cells expressing the full-
length receptor or the extracellular domain (ECD) anchored
via a glycophosphatidylinositol (GPI) link (3–10). Most stud-
ies report TSHR-binding autoantibodies in a proportion of
sera containing TSAB and/or TBII and demonstrate the
heterogeneity of TSHR autoantibodies, e.g. a GD patient with
monoclonal gammopathy, having antibodies that bound
to the TSHR in a Western blot, but were TBII and TSAB
negative (11).
In the case of the TSHR, antibodies without biological
effect may be of particular relevance in the extrathyroidal
manifestations of GD, thyroid eye disease (TED), and
pretibial myxedema. Pretibial myxedema is rare, but TED is
frequently associated with GD, and several lines of evidence
indicate that the TSHR may have a role in both disorders (12).
These include the demonstration of TSHR transcripts and
protein in the ocular tissues, especially in the adipose com-
partment (13–15), and the development of animal models of
TED induced by transfer of TSHR-primed T cells (16) or
TSHR genetic immunization (17). A strong argument against
the TSHR being involved in TED is the fact that not all
patients with TED have TSAB or TBII. This heterogeneous
Abbreviations: ECD, Extracellular domain; FITC, fluorescein isothio-
cyanate; GD, Graves’ disease; GPI, glycophosphatidylinositol; TBAB,
thyroid-blocking antibodies; TBII, TSH binding-inhibiting Igs; TED, thy-
roid eye disease; TPO, thyroperoxidase; TSAB, thyroid-stimulating an-
tibodies; TSHR, TSH receptor.
0013-7227/02/$15.00/0 The Journal of Clinical Endocrinology & Metabolism 87(4):1754 –1761
Printed in U.S.A. Copyright © 2002 by The Endocrine Society
1754
group of individuals with severe symptoms of TED in the
absence of TSHR autoantibodies in conventional assays,
which we have termed atypical TED, may have no evidence
of thyroid dysfunction or may have been thyrotoxic
previously.
TSAB are predominantly of the IgG1 isotype, and there is
some evidence for light chain restriction. Other isotypes have
been implicated in the extrathyroidal manifestations of GD
(18), and increases in the level of IgA secretion in tears have
also been reported in 25% of patients (19). Furthermore, in
the receptor-induced animal models of TED, mast cells were
an early indicator of orbital pathology, and elevated IgE
levels have been found in GD patients (20), both of which are
suggestive of IgE immunoreactivity to the TSHR.
The aim of the present study was to investigate antibodies
binding to the TSHR in individuals negative for TSAB and
TBII, either normal controls or patients with atypical TED,
and to determine whether TSHR autoantibodies are exclu-
sively of the IgG isotype and/or light chain restricted in these
and a panel of GD sera.
Subjects and Methods
Subjects studied
Serum samples from a total of 110 individuals were tested. This
included 65 untreated patients with GD, defined by thyrotoxicosis, a
diffuse goiter, and the presence of Tg or TPO antibodies or an appro-
priate family history or exophthalmos, and 12 patients with treated GD.
Of the latter group, 6 also had TED, i.e. exophthalmos measured by
exophthalmometry. All of these patients had TSHR autoantibodies de-
tected as TSAB or TBII. We also selected 8 atypical TED patients who
were negative for TSAB and TBII in conventional assays: 3 with no
evidence of thyroid dysfunction, 1 with newly diagnosed GD, and 4 who
were previously treated for GD. All 8 patients had exophthalmos of at
least grade II; their details are shown in Table 1. We also studied 25
biochemically euthyroid normal blood donors (16 females and 9 males)
selected at random from the local blood transfusion service. All samples
were obtained with informed consent and with the approval of the local
ethics committee in accordance with the Declaration of Helsinki.
Measurements of TSAB, TBAB, and TBII
TSAB and TBII were measured on CHO cell lines stably expressing
the human TSHR, lulu or NA-4 for TSAB (21, 22) and JP09 for TBII (23),
as previously described in detail.
Construction and characterization of GPI cell line
Construction of GPI-anchored TSHR ECD. A synthetic linker encoding a
thrombin cleavage site and an artificial GPI attachment site based on a
region of domain 3 of rat CD4 (7) was created by annealing of the
following seven oligonucleotides and cloned into the XhoI and XbaI sites
of pcDNA3.1 (Invitrogen, San Diego, CA), 5⬘-TCGAGCTGGTGCCAA-
GAGGCTCTATCGAGGGCAGA-3⬘,5⬘-CGTGATGGATGTGCCTCT-
GCCCTCGATAGAGCCTCTTGGCACCAGC-3⬘,5⬘-GGCACATCCAT-
CACGGCCTATAAGAGTGAG-3⬘,5⬘-CTCCGCTGACTCCCCCTCAC-
TCTTATAGGC-3⬘,5⬘-GGGGAGTCAGCGGAGTTCTTCTTCCTACTC-
3⬘,5⬘-CTAGCTAGACGAGCACGAGCAGGAGCAGAAGGATGAGT-
AG GAAGAAGAA-3⬘,5⬘-ATCCTTCTGCTCCTGCTCGTGCTCGTC-
TAG-3⬘,5⬘-GGCTCGAGTATGTCTTCACACGGGTT-GAACTC-3⬘, and
5⬘-GCCGGATCCATGAGGCCGGCGGA-CTTGCTG-3⬘.
The region coding the extracellular domain of human TSHR [amino
acids 1 (methionine) to 412 (isoleucine)] was amplified using the High
Fidelity Taq polymerase (Roche, Indianapolis, IN) and the following
oligonucleotide primers: sense, 5⬘-GCCGGATCCATGAGGCCGGCG-
GACTTGCTG-3⬘; antisense, 5⬘-GGCTCGAGTATGTCTTCACACGGG-
TTGAACTC-3⬘, and was cloned into the BamHI and XhoI sites upstream
of the GPI anchor sequence to give the plasmid pGPI-TSHR. The final
construct was verified by sequencing using an ABI 310 system (PE
Applied Biosystems, Foster City, CA). Plasmid pGPI-TSHR was trans-
fected into CHO-K1 cells using Tfx-50 (Promega Corp., Madison, WI)
according to the manufacturer’s instructions, and stable lines were se-
lected using G418 at a concentration of 400
g/ml. Clonal lines were
obtained and screened by FACS using TSHR monoclonal antibody 2C11
(Serotec, Oxford, UK).
The expression of TSHR ECD on the GPI cell surface was demon-
strated by flow cytometry using monoclonal antibodies A10 (1:50 dilu-
tion) (24), BA8 (1:10 dilution) (25), and 3G4 (1:50 dilution) (25), used as
culture medium, and 2
g/tube purified 2C11 (Serotec) to the human
TSHR, followed by an antimouse IgG-fluorescein isothiocyanate (Ig-
FITC) conjugate (1:32; DAKO Corp., Carpenteria, CA), as described in
detail below.
TSH binding studies were performed on 1.5 ⫻10
5
cells in 12-well
plates to estimate the numbers of receptors expressed at the surface of
the GPI cells and were compared with JP09 (and the control JP02, CHO
TABLE 1. ‘Atypical’TED patient details: summary of flow cytometry results
No./sex/age TSAB/TBII/TPO Treatment/diagnosis IgG IgA IgE
Euthyroid TED
P1 F 53 ⫺ve ⫺ve ⫹ve TED confirmed in all 3 cases by
C.T. and/or M.R.I. scan
⫺ve ⫺ve ⫺ve
P2 M 62 ⫺ve ⫺ve ⫺ve ⫺ve ⫺ve ⫺ve
P3 F 67 ⫺ve ⫺ve ⫺ve ⫹ve ⫹ve ⫺ve
GD with TED TSAB and TBII negative
P4 F 37 ⫺ve ⫺ve ⫺ve Previous MMI ⫹ve ⫺ve ⫺ve
P5 F 46 ⫺ve ⫺ve ⫹ve Previous PTU ⫹ve ⫹ve ⫺ve
P6 F 60 ⫺ve ⫺ve ⫺ve Previous MMI ⫺ve ⫹ve ⫺ve
P7 F 47 ⫺ve ⫺ve ⫺ve Radio-iodine ⫺ve ⫺ve ⫺ve
P8 F 32 ⫺ve ⫺ve ⫹ve Untreated ⫹ve ⫹ve ⫺ve
GD with TED TSAB positive
GD 1 F 60 ⫹ve ⫹ve ⫹ve Previous MMI ⫹ve ⫹ve ⫹ve
GD 14 F 62 ⫹ve ⫺ve ⫹ve Untreated ⫺ve ⫺ve ⫺ve
GD 15 F 49 ⫹ve ⫺ve ⫹ve Untreated ⫺ve ⫺ve ⫺ve
Normal subject
N6 F 52 ⫺ve ⫺ve N.D. ⫹ve ⫹ve N.D.
C.T., Computerized tomographic; M.R.I., magnetic resonance imaging; MMI, methimazole; PTU, propylthiouracil; N.D., not determined.
TSAB and TBII were measured on CHO cell lines stably expressing the human TSHR, lulu, or NA-4 for TSAB (21, 22) and JP09 for TBII
(23), as described previously, and TPO antibodies using a Roche Immunodiagnostics enzymun test, values greater than 32 kU/liter being positive.
Metcalfe et al. •IgG, IgA, and IgE TSHR Autoantibodies J Clin Endocrinol Metab, April 2002, 87(4):1754 –1761 1755
cells expressing the neomycin resistance plasmid but no TSHR). Binding
was performed in binding buffer comprised of NaCl-free Hanks’solu-
tion, 280 mmsucrose, and 0.2% BSA. Initially, saturation curves, using
increasing volumes of [
125
I]TSH (Brahms Diagnostica, Berlin, Germany)
from 50–500
l/well and an equal volume of binding buffer, were
carried out. Subsequently, the optimized volume of tracer was competed
for by increasing concentrations of cold bovine TSH (Sigma, St. Louis,
MO)for2hatroom temperature. Cells were washed twice with the
binding buffer and lysed with 0.5 ml 1 nNaOH/well, and bound
radioactivity was determined in a
␥
-counter. All measurements were
made in duplicate and performed at least three times to calculate average
EC
50
and binding capacity values from Scatchard analysis, reported as
milliunits per ml TSH.
Flow cytometry
Seventy to 90% confluent cells (GPI and JP02) were detached from
75-cm
2
culture dishes using 5 ml 5 mmEDTA and 5 mmEGTA in PBS.
The cells were washed three times in PBS containing 0.1% BSA and
adjusted to 2 ⫻10
6
cells/ml in the same buffer. Aliquots (100
l) of cells
were incubated with 2
l heat-inactivated test serum from the various
patient and control groups for1hatroom temperature. After three
washes in PBS-BSA, they were incubated on ice in the dark for 30 min
with antihuman IgA-FITC conjugate (1:32, from Sigma), antihuman IgE
(1:50, from Sigma; 1:50, from Serotec) or antihuman IgG (1:50, from
Sigma). They received an additional three washes in PBS-BSA and were
resuspended in 1 ml of the same buffer, but containing propidium iodide
FIG. 1. Characterization of the GPI cell line. A, Typical
profile of TSHR staining at the surface of GPI. The faint
trace shows the fluorescence intensity using an isotype
control monoclonal, and the bold trace shows the fluo-
rescence intensity with TSHR monoclonal 2C11. The D
value is 0.93. B, TSH binding to TSHR expressing CHO
GPI and JP09 and the control JP02 cell lines. Binding
was performed with 40,000 counts/well (JP09 and JP02)
or 80,000 counts/well (GPI). The x-axis shows the con-
centration of added cold TSH in milliunits per ml, and
the y-axis shows the total counts bound (mean of du-
plicates agreeing to within 10%).
1756 J Clin Endocrinol Metab, April 2002, 87(4):1754 –1761 Metcalfe et al. •IgG, IgA, and IgE TSHR Autoantibodies
for gating out dead cells. Cells were also analyzed in the same protocol,
but omitting the first antibody (either TSHR monoclonal or patients’
serum) to control for nonspecific binding of the mouse and human FITC
conjugates. In addition, 12 of the samples positive for IgG binding to GPI
were analyzed further by replacing the antihuman IgG-FITC with either
an antihuman
or an antihuman
light chain-FITC.
Flow cytometric analysis was performed on a FACS Vantage from
Becton Dickinson and Co. (Mountain View, CA), incorporating a Co-
herent Enterprise II laser emitting at 488 nm. Forward light scatter, 90°
light scatter, and fluorescence emissions were collected for 1 ⫻10
4
cells,
and the geometric mean fluorescence intensity values of GPI and JP02
were compared for all sera, including the normal samples. In addition,
the Kolmogorov-Smirnov (K-S) two-sample test, which gives the great-
est difference between the two histograms (GPI and JP02) and is quoted
as the D value, was used (26), and cut-off values were defined based on
the mean ⫾2sd of the normal sera.
Results
Characterization of the GPI cell line
Surface expression of TSHR ECD was confirmed by flow
cytometry. Background staining with an isotype-matched
control antibody showed a median fluorescence of 4.03 and
peak channel fluorescence of 3 compared with 667 and 889
when using the 2C11 monoclonal (D ⫽0.93). Similar results
were obtained with the other three TSHR monoclonal anti-
bodies tested, which together recognize epitopes at the ex-
treme N- and C-termini of the TSHR ECD and the native
conformation of the human TSHR. A typical experiment is
shown in Fig. 1A.
Saturation curves for [
125
I]TSH binding demonstrated that
JP09 reached saturation with 150
l (40,000 cpm) tracer, but
GPI required 300
l (80,000 cpm) to achieve this, the first
indication of the greater surface receptor expression in the
latter cells (data not shown). TSH binding curves are shown
in Fig. 1B. The affinity of the ECD TSHR expressed in the GPI
cells is similar to that of the full-length TSHR in JP09, with
EC
50
values of 4.3 (range, 2–6) and 3.4 (range, 3–5) mU/ml
TSH, respectively. Scatchard analysis of the GPI and JP09 cell
lines produced Bmax values of 76 and 19 mU/ml TSH re-
spectively, indicating approximately 4 times as many recep-
tors expressed at the surface of GPI compared with JP09.
Flow cytometry
Each patient or control serum was assayed against both the
JP02 and GPI cell lines, because of variable reactivity of
human sera with the control receptor negative cell line, e.g.
when detecting IgG binding the geometric mean ranged from
14–92. The distributions of fluorescence and background
fluorescence were similar for the receptor-negative and -pos-
itive populations.
Based on the mean ⫾2sd of the normal control serum, a
D value of more than 0.28 was considered positive, and 55
of 65 untreated GD sera displayed specific positive binding
to the GPI cell population. As shown in Fig. 2, there was a
poor correlation (r ⫽0.22) between autoantibodies binding
directly to the TSHR and TSAB measured in a luminescent
bioassay.
Twelve of the sera positive on GPI were tested for
or
light chain restriction in their TSHR binding antibodies.
From the mean ⫾2sd of the 12 sera and the difference in
D value between
and
, 1 sample displayed complete
restriction, and 7 of the other 11 displayed more binding
associated with
than
light chains, as summarized in
Table 2.
Positive specific IgG binding to the ECD TSHR of the GPI
cells was also observed in 3 of 25 normal and 4 of 8 of the
TSAB/TBII-negative TED sera. Representative histograms
are shown in Fig. 3.
IgA binding to the control receptor-negative cells had a
range of geometric mean from 48 –112. A D value greater than
0.35 was taken as the cut-off, and positive IgA receptor au-
toantibodies were demonstrated in 1 of 12 normal sera, 1 of
7 GD with TED, and 3 of 8 of the TSAB/TBII-negative TED
sera. Representative results are shown in Fig. 4.
Circulating IgE levels are considerably lower than those
for IgG and IgA, making the production of specific antisera
more difficult. We applied two different IgE-FITC conju-
gates, both polyclonal antibodies raised in goat that pro-
duced cut-offs for the D values of 0.41 (Sigma) and 0.25
(Serotec), respectively. Positive IgE binding was demon-
strated, using both IgE-FITC conjugates, in 1 of 12 normal
sera (28-yr-old male), 2 of 6 GD without TED, 1 of 8 GD with
TED, and 0 of 8 atypical TED sera. Representative results are
shown in Fig. 5.
FIG. 2. TSHR autoantibodies in 65 patients with untreated GD.
TSAB were assessed by luminescent bioassay, and results are re-
ported as a percentage of a TSAB standard on the x-axis. Antibodies
(IgG) binding directly to the TSHR were measured by flow cytometry
on GPI cells, and results are reported as a D value on the y-axis. There
is a poor correlation (r ⫽0.22) between the two methods. The dotted
lines depict the normal cut-off (TSAB, ⬎40%; D ⫽⬎0.28) for TSAB
and direct TSHR binding, respectively.
TABLE 2. Lambda light chain predominance in direct binding
IgGs to the TSHR
GD sample ‘D’value kappa ‘D’value lambda
GD 2 0.51 0.81
GD 3 0.26 0.65
GD 4 0.44 0.60
GD 5 0.55 0.39
GD 6 0.55 0.53
GD 7 0.57 0.83
GD 8 0.55 0.46
GD 9 0.46 0.77
GD 10 0.42 0.55
GD 11 0.53 0.74
GD 12 0.69 0.67
GD 13 0.49 0.74
Based on the mean ⫹2SD of the group as a whole, D values for
kappa ⬍0.3 and for lambda ⬍0.37 were considered negative. On this
basis, patient 3 displays complete lambda restriction because the
kappa value is negative.
Metcalfe et al. •IgG, IgA, and IgE TSHR Autoantibodies J Clin Endocrinol Metab, April 2002, 87(4):1754 –1761 1757
Flow results for IgG, IgA, and IgE binding, showing the
presence of receptor antibodies of more than one isotype in
some individuals, are summarized in Table 1.
Discussion
We have produced a CHO cell line stably expressing the
ECD of the TSHR linked to the cell surface via a GPI anchor.
The characteristics of the GPI line are similar to those in two
earlier reports (7, 8) and indicate that the ECD has an affinity
for TSH comparable to that of the full-length TSHR and is
sufficient to detect binding of TSHR autoantibodies.
We have demonstrated IgG antibodies binding directly to
the receptor in the majority of untreated patients with GD,
in agreement with some (8, 10), but not all (9), previous
studies that have applied flow cytometric analysis. We found
a poor correlation between these direct binding antibodies
and TSAB, as reported by others (10). Our data lend further
support to the concept that TSAB comprise a small propor-
tion of TSHR autoantibodies. The results also indicate that
TSHR autoantibodies in general display a preference for the
IgG
isotype and that it is not a feature confined to TSAB (1).
The presence of TSHR autoantibodies in GD is not sur-
prising, but we have also obtained positive binding of IgG
and/or IgA antibodies to GPI cells in 50% of patients with
atypical TED and a small proportion of normal euthyroid
controls, all of them women. Binding of IgE antibodies to the
GPI cells, apart from 3 of 14 GD sera, was detected only in
one 28-yr-old normal male subject and in none of the TSAB/
TBII-negative TED patients.
The etiopathogenesis of TED remains a puzzle to endo-
crinologists, although most of the signs and symptoms can
be attributed to an increase in orbital volume as the conse-
quence of glycosaminoglycans production, edema, and fat
hypertrophy. A variety of immune cells and cytokines are
present in TED orbits, and TSHR-specific T cell lines have
been reported (27). Several additional lines of evidence favor
the TSHR as a common antigen in GD and TED, as described
above. The present study has demonstrated immunoreac-
tivity to the TSHR in a subset of patients with TED previously
assumed to be free of receptor autoimmunity. Their euthy-
roid state is explained by the neutral nature of their TSHR
autoantibodies, which lack TSAB or TBII activity. Given the
size of an Ig, it is perhaps surprising that an antibody that is
FIG. 3. IgG autoantibodies binding directly to the TSHR. Flow cytometry with fluorescence intensity shown on the x-axis and cell counts on
the y-axis. The bold trace was obtained from GPI cells, and the faint trace was obtained from JP02 receptor-negative control cells. A, Atypical
TED P4 (D ⫽0.5); B, atypical TED P1 (D ⫽0.2); C, normal 5 (D ⫽0.44); D, normal 7 (D ⫽0.19). A D value more than 0.28 is considered positive.
1758 J Clin Endocrinol Metab, April 2002, 87(4):1754 –1761 Metcalfe et al. •IgG, IgA, and IgE TSHR Autoantibodies
able to bind the receptor does not inhibit TSH binding, al-
though this has been reported previously (4), as have TSAB
that lack TBII activity (11). Measurement of direct binding
TSHR antibodies could be applied to confirm the diagnosis
if TED is suspected in an individual negative for TSAB and
TBII.
There are a few reports indicating TSHR autoreactivity in
normal healthy subjects, including the demonstration of re-
ceptor-specific T cell lines (28) and peripheral blood mono-
nuclear cells (29). More recently Atger and colleagues (3)
applied an ELISA, using a solubilized TSHR preparation to
coat the plates, and found neutral antibodies in a surprisingly
high proportion of euthyroid controls. In our series of normal
subjects, TSHR antibodies were confined to three women,
aged 46–52 yr, one of whom displayed both IgG and IgA
reactivity.
The idea of autoantibodies in healthy subjects is not novel,
and the other major thyroid autoantigens, Tg and TPO, are
associated with incidences of approximately 15% and 10%,
respectively; they are highest among middle-aged women
(2). It seems that the TSHR is not so different, although
studies using a larger panel of euthyroid male and female
subjects spanning all age ranges are required to determine
the prevalence of TSHR autoreactivity in the absence of
disease.
GD is very common, and our data imply that immuno-
reactivity to the target antigen may also be common. Is its
immunogenicity due to molecular mimicry of the TSHR by
structures on the surface of commensal bacteria, or does
receptor processing release/unmask an immunodominant
epitope? Immunodominant epitopes have been implicated in
a variety of autoimmune diseases, and indeed, there is a
suggestion that the development of thyroiditis requires re-
moval of immunodominant regions in a receptor-induced
animal model (30).
We were able to detect IgE immunoreactivity to the
receptor by flow cytometry in patients with GD and TED.
Interest in this arm of the immune response has been
kindled by an animal model of TED induced by passive
transfer of TSHR-primed T cells, which one of us has
reported (16). In this model, mast cell infiltration was one
of the earliest indicators of orbital pathology. Develop-
ment of TSHR autoantibodies of the IgE subclass by iso-
type switching would provide a neat explanation for the
severe TED found in some, but not all, GD patients. Re-
cently, elevated IgE levels have been reported in GD pa-
tients, although whether the antibodies were directed to
the TSHR was not investigated (20), although IgE anti-
bodies to another major thyroid autoantigen, TPO, have
been reported (31). Further studies, using a larger series of
FIG. 4. IgA autoantibodies binding directly to the TSHR. Flow cytometry with fluorescence intensity shown on the x-axis and cell counts on
the y-axis. The bold trace was obtained from GPI cells, and the faint trace was obtained from JP02 receptor-negative control cells. A, GD with
TED GD1 (D ⫽0.38); B, atypical TED P8 (D ⫽0.35); C, normal 6 (D ⫽0.72); D, normal 7 (D ⫽0.15). A D value more than 0.35 is considered
positive.
Metcalfe et al. •IgG, IgA, and IgE TSHR Autoantibodies J Clin Endocrinol Metab, April 2002, 87(4):1754 –1761 1759
patients, particularly GD patients before the onset of TED,
are warranted to resolve the issue of IgE antibodies to the
receptor.
In conclusion, we report neutral IgG antibodies, predom-
inantly of the
class, in the majority of patients with un-
treated GD and in a small proportion of middle-aged eu-
thyroid female controls. We have clearly demonstrated IgG
and/or IgA antibodies recognizing receptor conformation in
a high proportion of patients with TED who are negative for
TSHR autoantibodies with biological activity measured as
TSAB or TBII.
Acknowledgments
We are grateful to Drs. Costagliola, Banga, and Prabhakar for kindly
donating antibodies to the TSHR, and to Drs. Parkes and Lazarus for
providing patients’sera.
Received September 25, 2001. Accepted January 8, 2002.
Address all correspondence and requests for reprints to: Dr. M.
Ludgate, Department of Medicine, University of Wales College of Med-
icine, Heath Park, Cardiff, United Kingdom CF14 4XN. E-mail:
ludgate@cf.ac.uk.
This work was supported in part by Brahms Diagnostica GmbH
(Berlin, Germany) and grants from the Wales Office of Research and
Development and the United Kingdom Medical Research Council.
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FIG. 5. IgE autoantibodies binding directly to the TSHR. Flow cy-
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A, normal 15 (D ⫽0.29); B, GD with TED GD1 (D ⫽0.56). A D value
more than 0.41 is considered positive.
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FIRST WORLD CONGRESS: HORMONAL AND GENETIC BASIS OF
SEXUAL DIFFERENTIATION DISORDERS
May 17–18, 2002
Tempe, Arizona
Participants: Maria I. New (Chairman), New York, NY; Jean Wilson (Chairman), Dallas, TX; Richard
Behringer, Houston, TX; Camerino Giovanna, Pavia, Italy; Cheryl Chase, San Francisco, CA; Patricia
Donahoe, Boston, MA; Maguelone Forest, Lyon, France; James Griffin, Dallas, TX; Melvin Grumbach, San
Francisco, CA; Nathalie Josso, Montrouge, France; Ursula Kuhnle-Krahl, Munich, Germany; Robin Lovell-
Badge, London, UK; Berenice Mendonca, Sao Paolo, Brazil; Heino Meyer-Bahlburg, New York, NY; Claude
Migeon, Baltimore, MD; Dix Poppas, New York, NY; Martin Ritzen, Stockholm, Sweden; Jacques Simard,
Quebec, Canada; Garry Warne, Victoria, Australia; Amy Wisniewsky, Baltimore, MD.
The Conference lectures will include: Genetics of Sex, Anti-Mu¨llerian Hormone Mutations, Animal Models
of Intersex, Normal Sexual Differentiation, Ambiguous Genitalia in Humans, Hormones and Sexual Be-
havior, Rationale for Gender Assignment, Legal Aspects of Gender Assignment, Ethical Issues in Gender
Assignment, Role of Advocacy Groups, Prenatal Therapy in Congenital Adrenal Hyperplasia, Surgical
Reconstruction in Patients with CAH and Ambiguous Genitalia, Medical Management at Puberty, Questions
Frequently Asked by Patients and Their Families, Support Groups for CAH and Androgen Insensitivity
Syndrome, Outcome Studies in CAH, Choosing Gender in the Indian Cultural Context, Partial Androgen
Insensitivity Syndrome and Partial Gonadal Dysgenesis, Complete AIS and Congenital Micropenis, Males
with 17

-Hydroxysteroid Dehydrogenase Deficiency, 5
␣
-Reductase Deficiency, True Hermaphrodites.
For Congress registration and scientific information, please contact: Maria New, M.D., Professor and
Chairman, Department of Pediatrics New York Presbyterian Hospital, Weill Cornell Medical College, 525
E. 68th Street, New York, NY 10021. E-mail: minew@med.cornell.edu; Phone: (212) 746-3450.
This conference was previously scheduled to occur in Gubbio, Italy, on September 12, 2001, as “Update on
Androgen Disorders,”but was postponed due to the September 11 disaster.
Metcalfe et al. •IgG, IgA, and IgE TSHR Autoantibodies J Clin Endocrinol Metab, April 2002, 87(4):1754 –1761 1761
