GASTROENTEROLOGY

1998;115:13171321

RAPID COMMUNICATIONS
Autoantibodies to Tissue Transglutaminase as
Predictors of Celiac Disease
WALBURGA DIETERICH,*, EBERHARDT LAAG,* HEIKE SCHO PPER,*,
UMBERTO VOLTA, ANNE FERGUSON,I HELEN GILLETT,I ERNST OTTO
RIECKEN,* and DETLEF SCHUPPAN*,
*Department of Gastroenterology, Klinikum Benjamin Franklin, Free University of Berlin, Berlin, Germany; Medizinische
Klinik I, University of Erlangen-Nurnberg, Erlangen, Germany; Department of Internal Medicine, Cardioangiology, and
Hepatology, Policlinico S. Orsola, University of Bologna, Bologna, Italy; and IDepartment of Medicine, Western General
Hospital, University of Edinburgh, Edinburgh, Scotland

See editorial on page 1584.

Background & Aims: Immunoglobulin A (IgA) autoantibodies to endomysium (EMA) are highly specific and
sensitive markers for celiac disease. Recently, we
identified tissue transglutaminase (tTG) as the major if
not sole endomysial autoantigen. Methods: An
enzyme- linked immunosorbent assay (ELISA) was
established to measure IgA anti-tTG titers in serum
samples from
106 celiac patients with partial or subtotal villous
atrophy, 43 celiac patients on a gluten-free diet, and
114 diseased and healthy controls. Results were
corre- lated with clinical and histological data and
with EMA titers. Results: In patients with biopsyproven celiac disease consuming a normal, glutencontaining diet, 98.1% of the serum samples had
elevated IgA titers against tTG, whereas 94.7% of
the control sera were negative. IgA anti-tTG
correlated positively with semi- quantitative IgA EMA
titers (r = 0.862; P F 0.0001 ). Conclusions: An
ELISA based on tTGallows diagnosis of celiac
disease with a high sensitivity and specificity. IgA
anti-tTGand IgA EMA show an excellent correlation,
further confirming the enzyme as the celiac disease
autoantigen. Because the assay is quantitative, not
subjected to interobserver variation, and easy to perform, it will be a useful tool for population screening of
a hitherto underdiagnosed disease.

eliac disease is an enteropathy that is

characterized by small intestinal
lesions of variable severity. In geneti-

cally predisposed individuals, the disease is

triggered by ingestion of gluten, but its
manifestation seems to

GASTROENTEROLOGY

ease.37 Although celiac disease is

effectively treated by gluten withdrawal,
the tight association of EMA with the
disorder suggests the importance of
autoimmunity in the pathogenesis.8
We
recently
identified
tissue
transglutaminase (tTG) as the main if not
sole endomysial autoantigen in celiac
disease.9 This enzyme is synthesized by a
broad spectrum of cell types but is
usually
retained
in
intracellular
compartments. Upon wounding, tTG can
be released from cells, where it is
thought to aid in tissue repair by crosslinking extracellular proteins,10,11 and to
activate
transforming growth factor
12,13
1),
which enhances collagen
synthesis14
and
induces
intestinal
epithelial cell differentiation.15 After
identifying
tTG
as
the
putative
endomysial autoantigen of celiac disease,

1998;115:13171321
we wanted to
develop and validate an
enzyme-linked
immunosorbent
assay
(ELISA) that allows a reproducible,
sensitive, and specific screening of large
populations, in particular because recent
studies suggest a high prevalence of celiac
disease that may reach 0.3%0.4%16,17
and because intervals from first symptoms
to diagnosis may last many years.18

Materials and Methods

Patients
A total of 106 serum samples from
untreated celiac patients (71 female and 35
male) were examined. The patients had
different clinical and histopathologic degrees
of severity (malabsorption or partial or
subtotal villous atrophy) and responded with
clinical improvement to gluten withdrawal.
The mean age was 32.6 years, ranging from 4
to 80 years. In addition, serum samples from
43 celiac patients (34 female and

require additional external factors.1,2 Celiac disease is

Abbreviations used in this paper: ELISA, enzyme-linked
diagnosed by small bowel biopsy, but
immunosorbent assay; EMA, endomysial antibody; tTG, tissue
serum
immunoglobulin
A
(IgA)
transglutami- nase.
autoantibodies to endomysium (EMA), an
1998 by the American Gastroenterological
extracellular
constituent
of
smooth
Association 0016 -5085 /98/$3.00
muscle, show an almost 100% sensitivity
and specificity for celiac dis-

1318

DIETERICH ET AL.

GASTROENTEROLOGY Vol. 115, No. 6

Table 1. Number, Sex, and Age of Celiac

Patients on a Normal Diet, Celiacs on
a Gluten-Free Diet, and Controls

Celiacs, biopsyproven Celiacs on a

gluten-free
diet
Controlsa
Healthy controls
Inflammatory
bowel
disease
Primary biliar y
cir- rhosis
Lupus
erythematosus
Cystic fibrosis
Rheumatoid
arthritis
Metastatic gastric
cancer
Various diseases b

Age,

Ag
e,

No. of
patien
ts

Sex
(F/M
)

rang
e (
yr )

mea
n(
yr )

106

71/35

480

32.6

43
114
41

34/9
74/40

779
174

34.3
36.3

36
10
8
3
2
2
12

NOTE. Celiac patients on a gluten-free diet did not undergo

biopsy.
a
In the control group, biopsy specimens were available only
from 10 healthy control patients.
b
Included 1 patient each with infectious diarrhea,
abdominal pain, anemia, unclear diarrhea, small bowel
carcinoma, primary liver carcinoma, osteomyelofibrosis,
pancreatic carcinoma, T-cell lymphoma, familial
adenomatous polyposis coli, esophageal cancer, and
hepatitis C.

9 male; age, 779 years; mean age, 34.3

years) consuming a gluten-free diet for 612
months were studied (Table 1).
One hundred fourteen patients or controls
(74 female and 40 male; age, 174 years;
mean
age,
36.3 years)
with
various
unrelated diseases or no disease served as
controls.
They
included
patients
with
inflammatory bowel diseases, primary biliary
cirrhosis, lupus erythematosus, and a variety
of other inflammatory or neoplastic diseases
(Table 1).
IgA anti-EMA titers, assessed by indirect
immunofluores- cence on monkey esophagal
tissue slides in two reference laboratories
(A.F./H.G. and U.V.), were semiquantified as
follows: 0, not detectable; 1, positive at
dilutions between 1/5 and 1/20; 2, positive

hour at room temperature. After three washes,

the wells were incubated with 100 L of
peroxidase-conjugated rabbit anti- human IgA
(Dianova, Hamburg, Germany), diluted 1/1000,
in the same buffer. Unbound antibodies were
removed, and color
was developed by addition of 200 L 0.1 mol/L
sodium citrate,
1 mg/mL o-phenylenediamine hydrochloride
(Sigma), and 0.06% H2O2 (Merck, Germany),
pH 4.2, at room temperature for 30 minutes in
the dark. Absorbances were read on an ELISA
reader (MRX; Dynatech, Denkendorf, Germany)
at 450 nm.
All serum samples were initially tested in
duplicate at a dilution of 1/25. In case of
highly elevated titers (optical density [OD]
values >2.5), sera were further diluted to
obtain OD values in the linear range between
0.5 and 2.5 OD. After subtraction of the
background (normally <0.08 OD) the OD
values were multiplied with the serum
dilution to obtain the ELISA titers. With the
inclusion of 3 control serum samples in each
assay, intra-assay and interassay variations
were below 2.8% and 20% (15 independent
tests), respectively.
between 1/40 and 1/80; 3, positive at 1/100;
4, positive at 1/200; and 5, positive at
dilutions
>1:200.

ELISA for tTG

Microtiter
plates
(96-well;
Greiner,
Frickenhausen, Germany) were coated with 1
g of tTG per well (single batch, 0.00167 U of
tTG activity) from guinea pig liver (Sigma,
Deisenhofen, Germany) in 100 L of 50 mmol/L
Tris-HCl, 150 mmol/L NaCl, and 5 mmol/L
CaCl 2, pH 7.5, for 2 hours at 37C with a
coating
efficiency
of
23.0%29.4%
as
determined by addition of a radiolabeled
tracer. Wells were extensively washed with 50
mmol/L Tris-HCl, 150 mmol/L NaCl, 10 mmol/L
EDTA, and 0.1% Tween 20, pH 7.4, and the
plates were incubated in washing buffer for at
least 10 minutes at room temperature or
overnight at 4C. Sera, diluted in 100 L of the
same buffer, were added to the wells and
incubated for 1

1318

DIETERICH ET AL.

Statistics
The Spearman correlation was used to
compare the stochastic EMA values with the
numerical ELISA titers; the KruskalWallis
test was used to compare untreated celiacs
with celiacs on a gluten-free diet or with
the controls. The sensitivity of the ELISA
was calculated as the frequency of positive
IgA anti-tTG titers in patients with biopsyproven,
EMA-positive
celiac
disease
consuming a normal diet, and the specificity
as the frequency of negative IgA anti-tTG
titers in nonceliacs without EMA, considering
a titer of 2:15 as a cutoff value, which
excluded 95% of the nonceliac patients.

Results
An ELISA based on serum IgA
autoantibodies
against
tTG
was
performed; the results are summarized
in Table 2. The ELISA procedure was
improved from the protocol used in our
previous study.9 Blocking with bovine

GASTROENTEROLOGY Vol. 115, No. 6

serum albumin was avoided because some

serum samples from patients with celiac
disease and controls showed antibodies
against this food component, therefore
possibly falsifying the results. Also, the
addition of calcium to the coating buffer
was mandatory to improve the sensitivity
of the ELISA because the antibodies of
celiacs reacted more strongly with the
calcium-activated form of tTG.
With a cutoff value of 2:15
(excluding 95% of
nonceliacs) to define a positive reaction
for circulating IgA antibodies to tTG, 4
patients with celiac disease were negative
for tTG but had a weak to moderate
positivity for EMA (Table 2). Ten serum
samples without detectable IgA EMA
(fluorescence score, 0) had elevated IgA
anti-tTG (ELISA titer 2:15; Table 2).
However, the number of false-positive IgA
anti-tTG results was re- duced to 6 when
1 patient with celiac small intestinal

December 1998

TRANSGLUTAMINASE ANTIBODIES IN CELIAC DISEASE 1319

Table 2. IgA Anti-tTG and Corresponding IgA

EMA Titers of 106 Celiacs, 43 Celiacs on
a Gluten-Free Diet, and 114 Controls
No. of
patien
ts
Untreated celiacs

1
6

21
35
28
15
Celiacs on a gluten-free 20
diet
7
12
2
1
1
Controls
114
40,

IgA
EMA
0
1
2
3
4
5
0
1
2
3
4
5
0

IgA anti-tTG
58
12, 17346
24758
7, 831253
763213
1766282
312, 27, 39, 115
1, 12, 1836
161402
83, 98
144
262
114, 19, 22, 31,
43, 129

NOTE. IgA anti-tTG titers are averages of duplicate

determinations, and EMA titers have been subdivided into
6 categories, ranging from no (0) to strong (5) reactivity.
The numbers for IgA anti-tTG represent single values or
the ranges of the titers. Sera that did not show
congruent IgA anti-tTG and EMA titers have been
highlighted: sera with negative IgA anti-tTG (<15) and
positive EMA (2:1) are marked by italic type, and sera
with positive IgA anti-tTG (2:15) and negative EMA (0) are
marked in bold type. For details, see text.

lesions (partial villous atrophy) and 3

celiacs on a gluten-free diet were
subtracted (Table 2). In patients with
biopsy-proven and EMA-positive disease,
the calcu- lated sensitivity and specificity
of the ELISA reached 98.1% and 94.7%,
respectively. The high sensitivity of the
ELISA
was
highlighted
by
the
examination of sera from 6 patients with
increased intraepithelial lympho- cytes,
an early mucosal change in celiac
disease.
These
patients,
also
characterized by short stature, abdominal
pain, constipation, recurrent miscarriage,
or irritable bowel syndrome, had low
EMA titers (score 1 in 5 patients; score 0
in 1 patient) and slightly increased IgA
anti-tTG titers, with 15, 16, 19, 36, 45,
and 72 (data not shown).
When the IgA titers to tTG were plotted
against the IgA EMA scores, a positive
correlation (r = 0.862; P < 0.0001 ) was
obtained (Figure 1 ). Additionally, the IgA
anti-tTG titers of untreated celiacs were

significantly increased compared with the

titers of celiacs on a gluten-free diet and
with controls (Figure 2).

Discussion
We used an ELISA based on tTG, the
recently
discovered
endomysial
autoantigen
in
celiac
disease,
to
determine (1) the extent to which
detectable IgA anti- tTG was predictive of
a
positive
EMA
test
result
by
immunofluorescent examination and (2) to
establish the

December 1998

Figure 1.
Semilogarithmic plot of the correlation
between IgA anti-tTG and EMA scores of 106 patients
with biopsy-proven celiac disease consuming a normal
diet, 43 celiac patients after a gluten-free diet, and 114
controls. The median (25/75 percentiles) of IgA antitTG relative to EMA were as follows: EMA (0), 4
(2/7); EMA (1), 18
(17/23); EMA (2), 105 (43/210); EMA (3), 180 (117/363);
EMA (4),
557 (191/762); and EMA (5), 637 (244/1193). r =
0.862; P <
0.0001.

titers of IgA anti-tTG in serum samples

from patients with celiac disease and
from various controls.
Collectively, IgA anti-tTG titers showed
a good corre- lation with EMA titers,
reaching a sensitivity of 98.1% and a
specificity of 94.7%. As predicted, IgA
anti-tTG were normal or significantly
lower in patients on a gluten-free diet
than in untreated celiacs. This is in line
with EMA titers that tend to disappear
after gluten withdrawal. In addition to
the intriguing implications that the

TRANSGLUTAMINASE ANTIBODIES IN CELIAC DISEASE 1319

discovery of tTG as the celiac disease

autoantigen
may
have
for
the
pathophysiology of the disease,1921 this
ELISA could replace the traditional
semiquantitative, observer-dependent, and
time-consuming indirect immu-

Figure 2. Semilogarithmic plot of IgA anti-tTG titers of

106 celiac patients on a normal diet (cd), 43 celiac
patients after a gluten-free diet (gfd), and of 114 controls
(ctr), with the following medians (25/75 percentiles): cd,
199 (118/671); cd/gfd, 18 (5/48); and ctr, 4 (2/7).
Differences between groups were significant (P < 0.001).

1320

DIETERICH ET AL.

nofluorescence test on sections of monkey

esophagus and may allow the screening
of large populations for celiac disease.
Four serum samples with positive IgA
EMA (2 at a dilution of 1/5 and 1 each at
dilutions of 1/10 and 1/100) showed no
detectable IgA anti-tTG even on repeated
testing. The endomysial antigen used for
these immuno- fluorescence tests was of
monkey origin, whereas the tTG used for
the ELISA is derived from guinea pig liver.
The
occasional
but
unequivocal
discrepancies between the results of the
two techniques may be explained either by
(minor) interspecies differences or by the
existence of other minor autoantigens in
the extracellular matrix.
In addition, sera may preferentially
react with anti- genic neoepitopes
generated by cross-linking of tTG with
gliadin or by potentiation of gliadin
antigenicity by deamidation.21,22 We
have shown a preferred cross- linking
of gliadin by tTG and an incorporation of
tTG itself in these complexes, as well as
IgA antibodies that are directed to these
cross-links rather than to tTG or gliadin
alone (Dieterich et al., unpublished
observations). Ten serum samples had no
detectable IgA EMA but an elevated IgA
anti-tTG titer (Table 2). Three of these
samples were from celiac patients on a
gluten-free diet and 1 from a patient
with partial villous atrophy, a finding
consistent with celiac disease. This
suggests that the sensitivity of the tTGELISA to detect celiac disease may be
greater than that of the indirect
immunofluores- cence method, without
significant loss of specificity. Because
small intestinal biopsy specimens had not
been obtained from the other 6 persons in
this group (3 with systemic lupus
erythematosus, 1 with primary biliary
cirrhosis, 1 with Crohns disease, and 1
without known disease), prospective
studies must clarify if such patients have
silent or latent celiac disease rather than
representing false-positive results.

GASTROENTEROLOGY Vol. 115, No. 6

Preliminary data on patients with IgA

deficiency, a condition with an at least 10fold increased incidence of celiac disease,
indicate either elevated IgA anti-tTG titers
despite low IgA levels or high levels of
IgG anti-tTG (Dieterich et al., unpublished
results). Because the IgG autoantibodies
may also be elevated in other inflammatory
or
autoimmune
diseases,9
the
predictive value of the IgG anti-tTG test in
these patients has to be confirmed in a
larger study.
In summary, our data on a spectrum of
patients confirm that tTG is the major if
not sole endomysial autoantigen of celiac
disease9 and suggest that an ELISA based
on this protein could be a useful tool to
screen large
populations
for
the
prevalence of this fairly common

1320

DIETERICH ET AL.

disorder, which might otherwise remain

undetected for many years.

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Received June 16, 1998 . Accepted July 31, 1998 .

Address requests for reprints to: Detlef Schuppan, M.D., Ph.D.,
Medizinische Klinik
I, University of Erlangen-Nu rnberg,
Kranken- hausstrasse 12, 91054 Erlangen, Germany. e-mail:
detlef. schuppan@med1.med.uni-erlangen.de; fax: (49) 9131
-8536003 .
Supported by grants SFB 366 C5 and Schu 646/4-1 from the
Deutsche Forschungsgesellschaft and by a grant from the German
Celiac Society.

Laennec of Laennecs cirrhosis

Rene Theophile Hyacinthe Laennec (17811826) was born at Quimper in Lower Brittany. The earl
Contributed by WILLIAM S. HAUBRICH, M.D.
Scripps Clinic and Research Foundation, La Jolla, California