Clinical Usefulness ofTSH Receptor Autoantibody Using Different

Clinical Usefulness ofTSH Receptor Autoantibody Using
Different Assay Systems
Yukari MIMURA, Toshio OGURA * and Fumio OTSUKA **
Abstract
Thyroid stimulating hormone receptor antibody (TRAb) plays an important role in
Graves' disease (GD). A second-generation measurement system has been developed
and we have gotten a benefit by the system clinically. In this study, we determined 4
kinds of TRAb in 42 GD patients using the current and second-generation
measurement systems to investigate the differences between them. The secondgeneration measurement system exhibited higher positive rates and inhibition rates of
thyroid stimulating hormone (TSH) binding than those of the current system.
Furthermore, 42 patients with GD were classified into 4 groups by GD activity. The
actual values of all TRAbs and positive rates exhibited a tendency to increase
significantly with GD activity. Of significance, 2 TRAbs in the second-generation
measurement system exhibited high positive rates. However, all actual values of
patients did not necessarily agree with these tendencies. The values of TRAb-human
detecting anti-human TSH receptors at an approximate cut-off value reflected GD
activity more accurately than those of TRAb-CT detecting anti-porcine TSH receptor.
This suggests the possibility of specific differences between TSH receptors and
further studies are required to further examine these effects.
Keywords: Graves' disease, human-TRAb, TSAb
Introduction
The TSH receptor is one of the major
autoantigens in autoimmune thyroid disease
and the pathogenetic role of autoantibodies to
the TSH receptor (TRAb) in sera from patients
with autoimmune hyperthyroidism has been
clearly established l ). Historically, there are two
established methods for the detection of
TRAb 2). One is the classical radioreceptor assay
of Smith3), based on the porcine TSH receptor,
where autoantibodies and labeled bovine TSH
compete for the binding sites of the receptor.
The other method is based on the ability of
some autoantibodies similar to TSH to induce
the second messenger cAMP. These bioassays
are able to distinguish between stimulating or
blocking autoantibodies, based on their
biological activity to either enhance or inhibit
the production of cAMp4). Although other
detection systems have been described, such as
autoantibody detection by FACS5),
immunocytochemistry6), or imrnunoprecipitation7),
these methods are still in an experimental state
Clinical Usefulness of TSH Receptor Autoantibody Using Different Assay Systems
Yukari MIMURA, Toshio OGURA * and Fumio OTSUKA * *
Division of Developmental Studies and Support, Okayama University Graduate School of Education, * Health and
Medical Center, Okayama University, * * Department of Medicine and Clinical Science, Okayama University
Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences
Division of Developmental Studies and Support, Okayama University Graduate School of Education 3-1-1
Tsushima-naka, Kita-ku, Okayama city 700-8530
- 135 -
Yukari MIMURA, Toshio OGURA, and Fumio OTSUKA
and are not available for routine commercial use.
In a recent report, Costagliola and co-workers8 )
demonstrate a second generation TSH binding
inhibitory assay using the human recombinant
TSH receptor with a significant gain in
sensitivity compared to the conventional
porcine antigen-based system. A new coatedtube radioreceptor assay for measurement of
TRAb using porcine TSH receptor has also
recently been developed9J • These assays have
been reported to exhibit high sensitivity in
untreated Graves' disease (GD) patients without
loss of specificity in healthy individuals 8 . 9).
However, the usefulness in GD with various
clinical states remains controversial. Then the
aim of this study was to evaluate the TRAb
assay for various clinical states of GD.
Subjects and methods
Patients
Serum samples from 42 patients with GD
were examined (mean age, 47.7 year old [range;
17-82]; 11 male and 31 female). These patients
attended our thyroid clinic from September to
October 2002. Among them, 5 were untreated
GD and others were GD that had been treated
for 2-360 months (88.4 ± 15.8, mean ± SD).
TSH
ATD Dosp
Change of TSH
TSAb
Group I ]
[ Group II
I
[Group III
I
[Group IV
I
Figure 1. Activity of GD according to TSH, ATD
dose, change of TSH, and TSAb.
Twenty-nine patients had taken thiamazole
(MMI) and 8 patients had taken propilthiouracil
(PTU). Patients were grouped according to
their state of GD, which was determined by the
serum TSH level, dose of antithyroid drug
(ATD) etc. (Fig 1). Briefly, all patients were
initially divided according to their serum TSH
levels, and patients whose TSH were
suppressed to the undetectable range, belonged
to Group 1. Patients with detectable TSH levels
were then sub-divided according to the daily
dose of ATD, and if they took over 3 tablet of
ATD (over 15mglday MMI or 150mglday PTU),
they belonged Group II. The change of serum
TSH (LlTSH) was then compared between the
last and present data. Those patients with
negative!'>TSH values also belonged to Group II.
Lastly, those patients positive or negative for
TSAb were sub-divided into Groups III or IV,
respectively. The groups are summarized in
Table 1.
Patients' samples were collected at the
department of endocrinology according to the
institutional guidelines for ethical conduct. All
samples were frozen immediately, free T3, free
T4 and TSH were measured by the ECLIA assay
system simultaneously, and stored at -20°C for
further assays.
TRAb detection
We measured anti-TSH receptor antibodies
(TRAb) from 4 different methods and thyroid
stimulating antibody (TSAb). Two TRAb
methods utilized the conventional radio
receptor assay with the porcine TSH receptor.
TRAb-D was measured using the TRAb DADE
(Dade Ltd, Tokyo, Japan) and TRAb-III was
measured using the Cosmic TRAb III (Cosmic
Ltd, Tokyo, Japan). The results were expressed
as the percentage inhibition of TSH binding.
The suggested cut-off was 15% and 10%,
respectively. Two additional TRAb methods
Table 1. Characteristic features in the patient groups.
Group n
I
12
II
15
III
8
IV
7
MJF Age (years)
1/11 36.4 ± 4.6
6/9
48.5 ± 4.8
4/4
54.6 ± 5.1
0/7
57.4 ± 5.5
TSH (pU/ml)
<0.01 ± 0.00
6.36 ± 1.89
3.95 ± 1.02
2.83 ± 0.37
IT3 (pglml)
6.54 ± 1.25
2.84 ± 0.16
2.94 ± 0.26
2.40 ± 0.08
- 136 -
IT4 (ngldl)
1.92 ± 0.29
0.91 ± 0.08
1.12 ± 0.13
1.15 ± 0.07
TSAb (%)
356 ± 68
316 ± 43
203 ± 7
125 ± 21
ATD Dose (tablets/day)
4.5 ±
2.8 ±
1.4 ±
1.1 ±
0.9
0.5
0.2
0.2
Clinical Usefulness of TSH Receptor Autoantibody Using Different Assay Systems
utilized second-generation assays for anti TSH
receptor antibodies in coated tubes. TRAb-CT
and TRAb-human were used to detect the
porcine and human recombinant TSH receptors,
respectively. These results were also expressed
as the percentage inhibition of TSH binding.
The suggested cut-off was 10% for each of these
procedures. Additionally, the TRAb-human
assay results were expressed in international
unit (IU) based on the WHO standard21 , with a
cut-off of 1.0 lUlL, and a "gray zone" of 1.0-1.5
lUlL. TRAb-CT and TRAb-human were
measured using the Cosmic TRAb CT (Cosmic
Ltd, Tokyo, Japan) and DYNOtest TRAK human
(BRAHMS AG, Berlin, Germany), respectively.
We also measured TSAb using the TSAb assay
kits (Yamasa Co., Tokyo, Japan). The suggested
cut-off for TSAb is 180%.
recommended cut-off values, are presented in
Table 2. Among the 4 TRAb assays, the data
from the TRAb-CT and TRAb-human assays
were higher than that of the TRAb-D (p<O.Ol).
The highest positive ratio was TRAb-CT with a
ratio of 73.8%. The lowest positive ratio was
TRAb-D.
Table 2. Serum autoantibodies related to
thyroid disease.
Assay kit
TRAb-D (%)
TRAb-III (%)
TRAb-human (%)
(lUlL)
TRAb-CT (%)
Data expressed mean ± SEM.
d~[ferent
The results of TRAb and positive ratio, which
were determined in accordance with the
A
~100
\00
o
80
Q
60
~ 80
~
----- _-_.
20
Eo-<
..
0
U
~
Ct::
_.
R=O.967
20
P<O.OOOl
•
·40
·60
Eo-<
40
20
SO
60
20
P<O.OOOl
•
0
100
~
40
Ct::
20
U
.-
20
40
so
60
100
~
P<O.OOOl
n=40
•
~
so
60
20
40
60
TRAb-D (%)
so
100
so
60
100
100
tR
' - ' 60
Eo-<
20
U
40
<
20
..6
Eo-<
f=
•
-40
0
40
,-.., so
40
·60
-20
20
F
-20
20
40
·20
TRAb-II1 (%)
100
R=O.888
·60
0
·20
20
Q
•
n=42
20
Eo-<
E
Eo-<
P<O.OOOl
40
~
n=42
R=O.893
60
-9
TRAb-II1 (%)
100
60
~
•
•
se
;:l
R=O.940
40
·20
D
Eo-<
e...-,100
60
TRAb-II1 (%)
80
-;J2
n=40
·20
,-..,
e.,
~120
'"
0
Eo-<
* p< 0.05, ** p< 0.01 vs TRAb-D.
C
B
40
Positive ratio (%)
30.0
57.1
57.1
61.9
73.8
4.3
4.0*
6.4**
4.0
4.5**
As shown in Figure 2, simple regression
analysis demonstrated that the % inhibition in
TRAb-human correlated positively with those of
TRAb-CT (r=0.965, p<O.OOOl; Fig. 2F), TRAb-III
(r=0.893, p<O.OOOl; Fig. 2C) and TRAb-D
(r=0.842, p<O.OOO1; Fig. 2E), respectively. These
findings in TRAb-CT also correlated positively
with those of TRAb-III (r=0.940, p<O.OOOl; Fig.
2B) and TRAb-D (r=0.888, p<O.OOOl; Fig. 2D),
respectively. The findings from TRAb-III also
Results
Detection TRAb and correlation between
assays
9.7 ±
24.0 ±
28.7 ±
11.2 ±
35.5 ±
·20
0
R=O.965
P<O.OOOl
11-42
0
·20
20
40
60
so
TRAb-human (%)
100
120
·20
a
20
40
60
SO
100
120
TRAb-hurnan (%)
Figure 2. Correlation between the two different TRAbs. (A) TRAb-O and TRAb-lIl, (8) TRAb-CT and
TRAb-llI, (C) TRAb-human and TRAb-llI, (0) TRAb-CT and TRAb-D, (E) TRAb-D and
TRAb-human, and (F) TRAb-CT and TRAb-human.
- 137 -
Yukari MIMURA, Toshio OG URA, and Fumio OTSUKA
TRAb in activity of CD
correlated positively with that of TRAb-D
~r=0.~67, p<O.OOOl; Fig. 2A). All 4 TRAb assays
exhibited significant correlations, and the
correlations were greater between TRAb-D and
TRAb-III or between TRAb-CT and TRAb-human. The strong correlation was thought to
be associated with the assay method.
TSAb in all patients was 274 ± 28%, and the
positive ratio was 69.0%. Positive correlations
were also evident between TSAb and the 4
methods of TRAb, but the correlatiuns were
weak compared with that between TRAbs
(r=0.495-0.648). The strongest correlation was
that between TSAb and TRAb-hwnan.
The summary of the TRAb assays in 42
patients is presented in Table 3. In those
patients with negative TRAb-CT (n= 11), the
other 3 TRAb assays were also negative. In the
patients with positive TRAb-D (n=13), the uther
3 TRAb assays were also positive. TRAb-D
exhibited the lowest detectiun rate, with only 10
negative patients. Among TRAb-III, TRAb-CT
and TRAb-human, there were 8 patients that
were positive in one or two TRAb assays. The
TRAb levels are shown below Table 3. Almost
all values were close to the cut-off value. Thus,
if the cut-off were to be altered, the positive
ratio would be different.
Table 3. Oiffereces in 4 assay of TRAb
positive
10.7
negative
6.2 - 9.3
positive
positive
positive
A
umber of cases
11
positive
10.2 - 21.5
1
25.2
1
T
positive
positive
11.6 - 32.1
positive
positive
positive
c
4
I
positive
positive
~ JTRA~b-~Il~I...\..o'--"-YoL-
I
1.6 - 3.6
B
..;.:::;.=--:"'----"-'-"--'-
TRAb-CT
l < 10%)
negative
TRAb-human
« 1.5 IUIL)
nf'gative
negative
0.9 - 1.4
negativp
1.47
TRAb-III
« 100AJ)
negative
negative
- 9.0 - 9.9
TRAb-D
« 15%)
negative
negative
- 51.3 - - 1.2
negative
- 5.2
negative
1.9 - 5.2
negative
3
T
1
10
14
D
__,
Gil
OJ
40
4.
10
-J-l--.il...--4--..........--.1 '"
-10
10
I
II III
IV
Activity of GD
1
II III
IV
Activity of GD
I
II III
IV
Activity of GD
I
*
II III
IV
Activity of GD
The rat io of
The ratio of
The ratio of
The ratio of
aboY<' the normal range (%) above the normal range (%) above the normal range (%) above the normal range (%)
loor
00
10
10
00
8
8
80
6
60
001
10
4
40
10
20
20
o
o
I
II
III
rv
Activity of GD
1
II
III
TV
Activity of (~ll
I
II
III
IV
Activity of GD
*p<O.05 and **p<O.O 1vs Group I
I
II
III
IV
Activity of GD
#p....:O.05 vs Group II
Figure 3. Level and positive ratio of (A) TRAb-D, (8) TRAb-lIl, (C) TRAb-CT, and (0) TRAb-human.
Data are presented as the mean + SEM. *p<O.05 and **p<O.01 vs Group I. #p<O.05 vs
Group II.
- B8 -
Clinical Usefulness of TSH Receptor Autoantibody Using Different Assay Systems
The various TRAbs according to GD activity
are shown in Figure 3. In all TRAb assays, the
levels of TRAb and positive ratio decreased
gradually according to the decline in activity of
GD. Groups III and IV were significant lower
compared with Group I. In Group I, in which
the patients were considered to be in an active
state of GD, both TRAb-human and TRAb-CT
exhibited a high positive ratio, 83.3%. On the
other hand, in Group IV, in which patients were
considered to be in a non-active state of GD,
although the TRAb-D and TRAb-human
exhibited a low positive ratio, 14.3%, the TRAbeT exhibited a high positive ratio, 57.1 %.
D-ifferencp between the TRAb
when the individual TRAb-III was 0%, are shown
in Figures 5A and 5B. Although the data of
TRAb--CT tended to increase, that of TRAbhuman tended to be distributed widely, from
negative to positive. We analyzed the difference
between TRAb-III and TRAb-CT or TRAbhuman according to the activity of GD (Fig. 5('
100
,--..
e
,§
The changes of individual data of different
TRAb are shown in Figure 4. The % inhibition
of TRAb-D, TRAb-III and TRAb-CT increased
gradually, but that of TRAb-human did not
exhibit this tendency, especially around the cutoff value. We then detected and further
examined 24 patients whose TRAb-III were
from 0 to 20%. The data, which was calculated
A
60
;D
40
ffif-<
20
c
0
:0
-:W
-3
assa~/s
80
c
.....0
~
-
~~~i-1 cut offvaJue
-40
-60 '--
---.J
TRAb-O TRAb-1II
TRAb-human TRAb·CT
Figure 4. Comparison of all TRAb in indibidual
subject. The lines represent the cut off
value. The cut off for TRAb-D was 15%,
and for TRAb-llI, TRAb-CT, and TRAbhuman the cut off valueswere 10%.
B
(%) 8 0 . . . - - - - - - - - - - ,
(%) 80
60
60
40
40
20
20
o I-~~~~~=---...,
-20 '-TRAb-III
Oi----iIiii
-=_--J
-20 ' - - - - - - - - - - - - - '
TRAb-1II
TRAb-CT
TRAb-human
C
D
(%) 30
(%) 30
20
20
10
10
--J
I
II
III
Activity of GD
IV
1
I
o
o r---l...l----LL----l...=;==j
-10 -'--
1
II
T
II
T
-10
I
II
III
IV
Activity of GD
Figure 5. Relative values of TRAb-human (A) and TRAb-CT (B) when the value of TRAb-11I was 0%.
Value of TRAb-human (C) and TRAb-CT (D) based on GD activity.
- 139 -
Yukari MIMURA, Toshio OGURA, and Fumio OTSUKA
and 5D). Both TRAb-CT and TRAb-human
decreased gradually according to the GD
activity, but the grade was greater for TRAbhuman compared with TRAb-CT.
Discussion
The TSH receptor is one of the major
autoantigens in autoimmune thyroid disease,
and autoantibodies acting as TSH receptor
agonists can lead to the clinical symptoms of
GDIO). There are two established principles
used to detect autoantibodies to the TSH
receptor 2l, and they are generally used to
diagnose G D, understand the severity of
disease, and to determine the effectiveness and
time to cease administration of ATD. Recently,
a new, second generation TSH binding
inhibitory assay system using coated tubes was
developed, that improved sensitivity without
loss of specificity in healthy individuals~J).
Furthermore, a full-length human recombinant
TSH receptor was developed and has been used
routinely in laboratories8, llJ. Several reports
have described the clinical usefulness of the
new assay systems, especially in the diagnosis
of G D2, 8, 9, 11-1;)). However, the relationship
between new TRAb assay and the severity of
GD has not yet been established. We therefore
evaluated the relationship between TRAb and
GD activity with 4 different TRAb assays and
TSAb simultaneously in GD patients.
Among the 4 assays of TRAb, the detection
rate was high for TRAb-CT, TRAb-human,
TRAb-III, and TRAb-D in all patients. The
detection rate of TRAb- CT and TRAb-human
was high compared with the other 2 methods in
Group I, which exhibited high GD activity, and
that of TRAb-CT was also high in Group IV,
which exhibited low GD activity. A high
positive ratio was reported, 98.3-100% in
untreated GD, 72.5-73.2% in treated GD, and
11.1-12.5% in healed GD9, 13). The reason for the
high sensitivity in the solid-phase assay was
ascribed to decrease non- specific binding
following washing of the tube, and the
prevention of contamination with TSH binding
agents, such as anti TSH antibodySl. In fact,
there was one present case with an anti TSH
antibody, for which level of TRAb-D was -51.3%
but the TRAb-CT and TRAb-human were
positive, suggesting that the influence of the
anti TSH antibody was negligible. A small
proportion (0.3-18%) of GD patients has been
reported to have an anti TSH antibodyI4-16\ so it
was important to measure the TRAb exactly and
not disregard this subset of patients. We can
take the very useful tool, on the contrary, we
were at a loss what to do. Since the patients
belong Group IV were formerly considered to
exhibit decreased activity, with negative TSAb
and low dose ATD (1.1 tablet/day), we could not
cease treatment with ATD, due to positive TRAb
findings.
We observed a fine correlation between the
individual TRAb assays (r>0.8), and the
correlation between TRAb-D and TRAb-III or
TRAb-CT and TRAb-human were stronger
(r>0.95). Since these two methods utilized
similar assay systems, respectively, we
considered that to be responsible for the high
correlation. There was also a strong correlation
between the solid-phase assay (TRAb-CT) and
the conventional method (TRAb-III) (r=0.940).
The correlation between the 4 TRAb assays and
TSAb were weak, as previously reported I7).
We attempted to examine the clinical data and
detection ratio in GD activity, by classifying the
patients into 4 groups. We classified the
patients according to serum TSH, the dose of
ATD, change of TSH compared to 2-3 months
ago, and TSAb. The TSAb and dose of ATD
gradually decreased in the four groups,
supporting the influence of G D activity,
although containing patients with transient
hypothyroidism following treatment with large
doses of ATD. In all assay methods, the
parameters and detection rate decreased
gradually, and the parameters were significantly
lower in Groups III and IV compared to Group I.
Although they were considered to be influenced
by GD activity, since the detection rate of TRAbCT and TRAb-human was higher in Group I, the
two new assay methods were useful to diagnose
GD. Second generation assays exhibited a high
- 140 -
Clinical Usefulness of TSH Receptor Autoantibody Using Different Assay Systems
detection rate in untreated GD, and were
effective in distinguishing GD from silent
thyroiditis9, 13, IS, 19).
There were 8 cases (19.0%) that exhibited
different results with the different assay
methods. Most of these were positive with
TRAb-CT or TRAb-human, and were negative
with the other assays. However, the titer was
almost at the cut-off level, so if the cut-off level
were to be changed, the detection would have
been changed. Kasagi also described the
estrangement of the results 20 ) due to similar
mechanisms. With treatment of GD, we were
able to observe the transition of the assay data,
and we were careful to administer ATD for a
long term. When reducing ATD, we should
consider not only TRAb, but also goiter size,
thyroidal radioactive iodine uptake and serum
Tg, in the decision-making process 13,21).
When we examined the individual data, TRAbCT, which used porcine TSH receptor, was
higher than TRAb-D and TRAb-III. However,
the data of TRAb-human, which used the human
recombinant TSH receptor, both increased and
decreased compared with TRAb-III. Especially,
the tendency was strong for the data around the
cut-off values with the conventional TRAb-III
methods, and we considered this to be greatly
influenced by GD activity. Although the TRAbhuman data is generally expressed in lUlL, we
used % inhibition to be able to compare the
results with the other assay methods. The %
inhibition was calculated without considering
non-specific binding (NSB) in the other 3
methods, but in this study we calculated the
data of TRAb-human considering NSB
according to the manufacturer's instructions.
We observed similar results even when we did
not account for NSB (data not shown). Some
reports have described that the species of THS
receptor did not impact the assay results, due to
the good correlation between the porcine and
human TRAb assaysS,22-24). However, the cloning
of the TSH receptor25-27) demonstrated that the
human and porcine TSH receptors were 85.0016
similar to in the extracellular domain, and 92.1%
in the transmembrane region 2S). In other words,
TSH in human is 15% different from porcine
TSH in the extracellular domain. Although this
difference appears to be minimal, researchers
still do not know the precise functional
conformation and the exact location of the
relevant TRAb epitopes2S). Further, differences
in the epitopes of TSAbs from GD patients were
apparent in untreated patients, and also after
antithyroid drug treatment29). So, although the
positive frequency of TRAb or TSAb was
similar, individual patients tend to exhibit
variability. Functional heterogeneity of TRAbs
has been reported 30). The TSH receptor is highly
homologous between species, such as bovine,
human, porcine and rat. However, following
TSH cloning of the human and porcine genes,
differences were evident on 15% in the
extracellular domain and 8% in the
transmembrane region 2S ). Some authors
described that TSH binding sensitivity was
improved when they used the human TSH
receptor3 1-34). The detection of various TRAb
with the human TSH receptor was useful and
improves the clinical relevance. In this study,
cases that were borderline using the
conventional TRAb assay were divided into two
groups for TRAb-human, the high clinical
activity group exhibited a high TRAb-human
titer and the low activity group exhibited a low
titer. We thought that this difference might be
due to the species of TSH receptor. Komori et aZ.
also described poor correlation in patients with
low TRAb-III titers (0-3OO16)1S). However, a larger
sample size of similar cases will be required to
validate these findings.
We hypothesized that these TRAbs could be
useful as an indication to stop ATD or relapse of
GD. However, we did not obtain the TRAb of
patients in Group IV that did not correlate one
year after thyroid function (data not shown).
Some authors have also reported that the use of
the second generation TRAb assays for the
precise prediction of relapse or remission in the
follow up of GD patients was questionable 35,36).
Since the remission and relapse of GD depend
on the size of thyroid gland, period of
medication, etc. 13, 21), we should consider when
the ATD was ceased.
In summary, new assay systems using the
- 141 -
Yukari MIMURA, Toshio OGURA, and Furnio OTSUKA
coated tubes were highly sensitive in G D
patients, and were influenced by GD activity.
Furthermore, TRAb-human exhibited some
specific advantages, which separated it from the
other assays.
Acknowledgment
We would like to thank Yamasa Co., and
Cosmic Co., for their technical support in the
study.
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