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Microbial transglutaminase used in bread preparation at standard bakery concentrations does not increase immuno-detectable amounts of deamidated gliadin Andreas Heil, Jürgen Ohsam, Bernard van Genugten, Oscar Diez, Keiichi Yokoyama, Yoshiyuki Kumazawa, Ralf Pasternack, and Martin Hils J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02414 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 20, 2017
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Journal of Agricultural and Food Chemistry
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Microbial transglutaminase used in bread preparation at standard bakery
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concentrations does not increase immuno-detectable amounts of deamidated
3
gliadin
4 5
Andreas Heil1, Jürgen Ohsam1, Bernard van Genugten2, Oscar Diez2, Keiichi
6
Yokoyama3, Yoshiyuki Kumazawa3, Ralf Pasternack1, Martin Hils1,*
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1
Zedira GmbH, Roesslerstrasse 83, Darmstadt, 64293, Germany
8
2
AB Enzymes GmbH, Feldbergstrasse 78, Darmstadt, 64293, Germany
9
3
Ajinomoto Co., Inc., Institute of Food Sciences & Technologies, 1-1, Suzuki-Cho,
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Kawasaki-Ku, Kawasaki-Shi, 2010-8681, Japan
11 12
* Corresponding author: phone +49 6151 325110; fax +49 6151 325119; e-mail:
13
[email protected] (M. Hils)
14 15
Conflict of interest statement
16
The authors declare the following competing financial interests: R.P., M.H., A.H., and
17
J.O. are shareholders and/or employees of Zedira. K.Y. and Y.K. are employees of
18
Ajinomoto. B.v.G. and O.D. are employees of AB Enzymes. Zedira received financial
19
support from Ajinomoto to perform this study. Ajinomoto did not participate in the
20
analysis and interpretation of the data.
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Abstract
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The effect of standard bakery concentrations of microbial transglutaminase (MTG) in
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wheat bread preparation on the immunoreactivity of celiac disease (CD) patients’
26
sera was investigated.
27
Immunoblotting using monoclonal
28
deamidated gliadin showed no differences between control bread and MTG-bread.
29
Deamidation of gliadin could not be detected at standard MTG concentrations.
30
CD patients’ sera were characterized using anti-gliadin and anti-DGP ELISA and
31
grouped into DGP high and low titer pools. The recognition pattern obtained after
32
using both CD sera pools for immunoblotting did not reveal differences between
33
control and MTG-treated bread protein extracts.
34
Our results indicate that MTG-treatment of wheat bread prepared with typical MTG
35
concentrations used in standard bakery processes does not lead to immuno-
36
detectable amounts of CD-immunotoxic deamidated gliadins.
antibodies
specific
to unmodified
37 38
Keywords:
39
microbial transglutaminase, celiac disease, gliadin, wheat bread
40
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Introduction
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Celiac disease (CD) is an autoimmune disorder triggered by the ingestion of gluten
43
derived from cereals like wheat, rye, or barley in susceptible individuals.1 The major
44
symptom of CD is chronic inflammation of the small intestine, combined with severe
45
damage of the intestinal mucosa. The wheat grain storage protein gluten is
46
composed of alcohol-soluble gliadins and propanol/urea/DTE-soluble glutenins.
47
Proteins present in the gliadin fraction, e.g. α- and γ-gliadins, are characterized by a
48
high content of proline. This renders gliadin resistant to proteolytic cleavage by
49
digestive enzymes of the gastro-intestinal tract. However, gliadins are broken down
50
into oligopeptides containing up to several dozen amino acids reaching the intestinal
51
mucosa. Another characteristic of these peptides is the high content of glutamine
52
residues in close proximity with prolins and hydrophobic residues, resulting in
53
multiple copies of the peptide motive QXPF in their sequence. This motif is a
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preferred substrate sequence for human tissue transglutaminase (TG2). Under the
55
specific conditions in the inflamed gut TG2 catalyzes deamidation of glutamines
56
instead of cross-linking, which is the general function of transglutaminases.2,3
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Deamidation results in a strong increase in affinity of the gliadin peptides towards
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HLA-DQ2/8 receptors present on antigen presenting cells. Binding of deamidated
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gliadin to HLA-DQ2/8 leads to CD4+ T helper 1 cells mediated intestinal
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inflammation.4 In addition it leads to the production of antibodies against deamidated
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gliadin peptides (DGP) as well as auto-antibodies against the endogenous TG2.2,5,6
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These (auto)antibodies are used as standard serological markers in CD
63
diagnostics 6,7, whereas anti-gliadin antibodies can also be related to other diseases.8
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The vicious circle of inflammation, transglutaminase release, deamidation of gliadin,
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and further immune stimulation currently can only be stopped by adhering to a
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completely gluten-free diet.9 3 ACS Paragon Plus Environment
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Microbial transglutaminase (MTG) is an enzyme derived from Streptomyces
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mobaraensis and catalyzes cross-linking of protein-bound glutamine to protein-bound
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lysine. Although it has a similar catalytic mechanism, it is not related to eukaryotic
70
transglutaminases in structure or sequence but is a result of convergent evolution.
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Most importantly, and in contrast to human transglutaminases, the microbial enzyme
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does not require Ca2+ for catalytic activity, thus enabling its industrial application.
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Under certain conditions, transglutaminases may also catalyze deamidation of
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protein-bound glutamine residues forming the respective glutamic acid. While TG2
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shows equal cross-linking and deamidation activities under the slightly acidic
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conditions as given in the duodenum this is not the case for MTG. Over a broad pH-
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range from 4 – 9 the deamidation activity of MTG constantly is more than a
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magnitude lower than its cross-linking tendency.10
79
The idea of a potential generation of CD-specific deamidated gliadin epitopes
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resulting from MTG-treatment of gluten-containing food was described in 2005.11 It is
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based on the hypothesis that MTG may mimic TG2 due to the similar mode of action.
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Since then this topic was further investigated by other research groups who found
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that MTG is able to deamidate gluten-related peptides which are immunotoxic in CD
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at least under certain experimental conditions.10,12,13 In contrast, other studies
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suggest that the use of MTG in bread preparations has no effect on the
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immunogenicity of gluten, but may even be used to detoxify gluten by covalent
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incorporation of primary amines.14,15 Despite of this, some research groups
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postulated the hypothesis that MTG used for production of baked products could lead
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to an increased incidence in celiac disease in Western countries.16–18 So far, to our
90
knowledge no clinical nor epidemiologic data is available whether there is a
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relationship between the apparent increase in CD and the use of MTG in baked
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goods.
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Commercial use of MTG
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Since its discovery in 1989, the use of MTG has spread into many different
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biotechnological applications, most importantly in food processing.19,20 The cross-
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linking function is used to improve properties of protein containing food like meat,
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sausage, fish, dairy products, pasta
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preparation was described using many different flour types, e.g. wheat, rye
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barley, soybean 24,25, oats 26, and mixtures of wheat and millet.27
21
and bread. The use of MTG in bread
28
22,23
,
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The use of MTG in bakery products was patented by Röhm GmbH in 1990
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was marketed by this company (nowadays known as AB Enzymes GmbH) for bakery
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related applications since approximately the mid 1990’s. First products were just
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enzyme preparations combining MTG with other enzyme classes. At a later stage a
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single activity MTG named VERON® TG (a product with 100 TG Units/g) was put on
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the market and happens to be the subject of this investigation. Since this product (like
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the majority of bakery enzymes available in the market) is based on a wheat flour
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carrier it is not suitable for the use in gluten-free bakery products. Therefore only a
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normal standard wheat bread application was considered for the purpose of this
109
study.
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Various articles and studies have been published showing the potential technological
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benefits of MTG in bakery applications.22,23,26,27,29–37 Generally, MTG has a positive
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effect on loaf volume when used at low concentrations, and at high concentrations
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the loaf volume is reduced due to formation of a strong meshwork of cross-linked
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proteins and therefore reduced expansion of gas bubbles.30–32
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However, typically MTG is never used as a single enzyme in a given bakery
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application but always in combination with other technologically required enzyme
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classes. Due to the limited field of benefits it can be considered a niche enzyme
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within the group of typical baking enzymes such as amylases, xylanases, lipases,
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oxidases, hemicellulases, cellulases, proteases, etc. and the global market share in
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terms of volume and value of MTG used for baking is estimated by industry experts
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to be lower than 1% of the total baking enzymes market.
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This low share is a result of its relatively low dosage (VERON® TG is used at 1-3 g /
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100 kg of flour, corresponding to 1-3 U/kg flour), that is a result of a relatively high
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cost in use and very specific functionality. Regardless of the low usage volumes MTG
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is sold and used for bakery applications on all continents without any significant focus
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countries. MTG is only required for solving explicit technical cases such as industrial
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bakers using wheat dough with short proofing times, where optimization of dough
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properties directly after mixing is crucial and MTG can complement the enzymes
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such as xylanase and amylase that are already used. This is the main reason why
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only low dosages of MTG (1-3U/kg flour) are required.
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Countries with traditionally high protein quantity and quality flours (such as derived
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from e.g. US or Canadian wheat) tend to have lower need for MTG use as compared
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to countries with predominant medium to poor quality flours, where MTG is used to
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improve dough elasticity to more closely resemble properties of high protein flour.38
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Currently in scientific journals as well as in the public, a discussion on a postulated
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celiac disease triggering effect by MTG takes place – controversial, due to lacking
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scientific data. Therefore, in order to provide a more solid base to assess this topic,
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the aim of this study was to investigate the effect of common industrially applied MTG
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concentrations in wheat bread production on the formation of deamidated gluten
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epitopes.
141 142 143
Materials and Methods
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Materials
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Microbial transglutaminase was applied as formulation VERON® TG (AB Enzymes,
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Darmstadt, Germany) containing MTG from Ajinomoto (Tokyo, Japan). Deamidated
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gliadin peptide DGPx2, 26mer gliadin peptide G051, as well as the monoclonal
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antibodies A011, A054, A064, A067 and A062 were from Zedira (Darmstadt,
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Germany). Celiac disease patients’ sera (n=26) were anonymized residual samples
150
purchased from in.vent Diagnostica (Hennigsdorf, Germany).
151 152
ELISA
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Celiac disease patients’ sera were analyzed for the following antibody titers (IgA and
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IgG) using commercial ELISA test kits according to the manufacturer’s instructions:
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gliadin (Steffens Biotechnische Analysen, Ebringen, Germany), DGP (Zedira,
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Darmstadt, Germany).
157 158
Bread preparation
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Bread was prepared according to an internal recipe by AB Enzymes (Darmstadt,
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Germany). Each bread contained 550 g wheat flour, 33 mg ascorbic acid, 11 g salt, 7 ACS Paragon Plus Environment
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11 g butter, 11 g sugar, and 27.5 g yeast. Three bread preparations contained
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different concentrations of VERON® TG (100 U/g): 20, 40, and 80 ppm,
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corresponding to a total of 2, 4 and 8 U MTG/kg flour. 2 U/kg flour is within the
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recommended dose range of 1 – 3 U/kg, 4 U/kg and 8 U/kg exceed the upper dose
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limit by up to 170%. The doughs were mixed in Diosna Spiral mixers type SP12
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applying standard mixing times of 2 minutes slow speed and 8 minutes fast speed to
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achieve an optimum dough development. The breads were fermented at 32°C and
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85% relative humidity for 80 minutes and then baked at 235°C for 35 minutes in
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unlidded baking tins.
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As positive control, bread was prepared according to Cabrera-Chávez et al.16 but
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with 2,000 U MTG/kg flour. For this MTG-concentration preliminary experiments
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revealed increased immuno-detectable deamidated gliadin peptides.
173 174
Preparation of albumin/globulin, gliadin and glutenin extracts
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Extraction was essentially performed as previously described.39 Briefly, 0.5 g bread
176
sample were added to 10 mL buffer consisting of 67 mM HKNaPO4, 400 mM NaCl,
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pH 7.6. Albumins and globulins were extracted at room temperature through vigorous
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mixing of the sample for 2 min, followed by gentle stirring for further 10 min and
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subsequent centrifugation at 6,000 x g for 15 min. Extraction of gliadins was
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performed using 60% ethanol (v/v). Glutenins were extracted at 60°C using 50 mM
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Tris-HCl pH 7.5, 50% (v/v) 1-propanol, 2 M urea, 1% (w/v) DTE.
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All extraction steps were performed three times with the same sample and the
183
supernatants from each step were pooled.
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Gel electrophoresis and immunoblotting
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SDS-PAGE was performed according to Laemmli.40 Briefly, samples were mixed with
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5x SDS-PAGE loading buffer (128 mM Tris-HCl pH 6.8, 10% β-mercaptoethanol,
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10% glycerol, 4% SDS, 13% bromophenol blue), boiled for 10 min and loaded on a
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10% SDS-PAGE gel. Separation was performed at 200 V for 40 min. Gels were
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stained with silver nitrate according to Blum et al.41 Electro-blotting was performed in
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a Trans Blot SD Semi Dry Transfer Cell (Bio-Rad, Hercules, U.S.A.) at 20 V for 80
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min. After blotting, the nitrocellulose membranes were presoaked in 48 mM Tris, 39
194
mM glycine, 1.3 mM SDS, 20% (v/v) methanol. Residual binding sites were blocked
195
in 5% skimmed milk powder in TBS-T (10 mM Tris, 150 mM NaCl, 0.05% (v/v)
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Tween 20, pH 8.0) for 60 min at RT. The membrane was washed in TBS-T wash
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buffer and incubated for 1 h at room temperature with primary antibody/serum
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(diluted 1,000 fold in TBS-T). After washing for 3 × 5 min in TBS-T, the secondary
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antibodies (anti-human IgA (Thermo Scientific, Karlsruhe, Germany), anti-human
200
IgG, anti-mouse IgG (both Sigma, Schnelldorf, Germany), each conjugated to
201
alkaline phosphatase and diluted 10,000 fold in TBS-T buffer) were added to the
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membrane followed by a 1 h incubation at room temperature. After intensive
203
washing, the membrane was placed in detection reagent (100 fold dilution of AP
204
color reagent in color development buffer, BioRad, Hercules, U.S.A.) and incubated
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for 30 to 60 s at room temperature. Excess detection reagent was drained off and the
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staining reaction was stopped with 20 mM Tris-HCl, 5 mM EDTA, pH 8.0. All washing
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steps were performed at room temperature on a shaker.
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Dot blots
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To analyze the specific binding epitopes of the antibodies used in this study, dot blot
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analysis was performed using unmodified or deamidated γ-gliadin 26mer (aa 59-84;
213
FLQPQQPFPQQPQQPYPQQPQQPFPQ
214
PEQPFPQ
215
LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF
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PELPYPQPELPY-PQPQPF [33mer DGP], respectively) fused to a carrier protein
217
(Zedira, Darmstadt, Germany). DGPx2 is a recombinant protein composed of both
218
33mer DGP and 26mer DGP fused to a carrier protein. Different amounts (1 and 10
219
µg) of either wheat gliadin (extracted from commercially available standard wheat
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flour) or recombinant protein were spotted on a nitrocellulose membrane and dried.
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The membrane was cut into small strips and each of them incubated with an
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individual antibody (A011, A054, A064, A057, A062). Further steps were identical to
223
the immunoblotting procedure already described.
[26mer
DGP],
and
respectively)
or
FLQPEQPFPEQPEQPYPEQα-gliadin and
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(aa
57-89;
LQLQPFPQPELPYPQ-
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Results and Discussion
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Determination of the specificity of monoclonal antibodies against wheat gliadin
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The monoclonal antibodies used in this study were evaluated for their specific binding
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epitope using dot blotting with different antigens containing either unmodified or
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deamidated immunotoxic CD epitopes, as well as wheat gliadin extracted from wheat
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flour. Figure 1 shows, that the antibodies strongly differed in their recognition pattern.
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A011 and A054 showed strong reactivity with wheat gliadin and recombinant
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unmodified 26mer γ-gliadin. A064 was able to recognize both unmodified and
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deamidated gliadin independent of the peptide sequence, which indicates that the
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binding epitope does not cover a deamidation site. Monoclonal antibodies A057 and
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A062
235
(KLQPFPQPELPYPQPQ).13 Whereas A057 is very specific for the deamidated
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peptide, A062 detects both unmodified and deamidated 33mer gliadin to a similar
237
extent. Therefore, A057 is one of the most important tools for identifying deamidated
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33mer gliadin peptide at the moment.
239
Importantly, none of the antibodies showed cross-reactivity with the carrier protein.
were
raised
against
the
same
immunogenic
peptide
240 241
242
Analysis of wheat gluten extracts from MTG-treated breads
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As expected baked breads prepared with increasing MTG-concentrations (2, 4 and 8
244
U MTG/kg flour) did not show visible differences, while high-dose positive control
245
MTG-bread (2,000 U/kg) was characterized by a small dough volume and reduced air
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bubble size. Albumin/globulin, gliadin and glutenin extracts from these breads were 11 ACS Paragon Plus Environment
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initially analyzed using SDS-PAGE followed by silver staining and Western blotting
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with an antibody detecting unmodified gliadin (A011) (Figure 2). Similar protein
249
amounts were extractable for albumin/globulin, gliadin, and glutenin fractions of the
250
control (0), 2, 4 and 8 U MTG/kg flour extracts. Western blot analysis revealed only
251
little gliadin detectable in the albumin/globulin extracts. As expected, more intensive
252
staining was observed for gliadin and glutenin extracts from the samples. The cross
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reactivity of anti-gliadin antibody A011 (as well as A064 and A054, see Figure 3) to
254
glutelins is in agreement with the observation by Martínez-Esteso et al. for anti-
255
gliadin antibodies used in gluten diagnostics.42
256
However, neither in silver stained gels nor in western blotting, differences in the band
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pattern between the 0, 2, 4 and 8 U MTG/kg samples were observed. An influence of
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MTG on the protein composition of the bread samples prepared with increasing
259
amounts of MTG could not be detected. This is in line with earlier data by Bauer et al.
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who, in a detailed analysis, did observe polymerization and decreased extractability
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of wheat flour proteins starting with 891 U MTG/kg flour. In contrast, with up to
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133 U MTG/kg no polymerization was detectable.43 Further the results are in
263
agreement with the observation for bread or pasta prepared with MTG (both about
264
300 U MTG/kg flour).16,39
265
In the positive control extract prepared with the extraordinarily high MTG-
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concentration of 2,000 U MTG/kg, high molecular weight bands are present
267
predominantly for gliadins and glutenins.
268
Further analysis (Figure 3) of the extracts was performed using additional monoclonal
269
antibodies recognizing unmodified gliadin (A054) or both unmodified and deamidated
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gliadin peptides (A064) (for specificity, see Figure 1). All samples showed intensive
271
staining with both antibodies. The staining in the albumin/globulin-fraction may be 12 ACS Paragon Plus Environment
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explained by residual amounts of gliadin. Again, and in agreement with the silver
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stained gels (Figure 2 A, B, C), an influence of MTG on the protein composition of the
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extracts prepared with increasing amounts of MTG could not be observed. We
275
already reported analogous results for pasta treated with 300 U MTG/kg.39
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Taken together, MTG-treatment of wheat dough only results in detectable amounts of
277
high molecular weight proteins, if MTG concentration is extraordinarily high (>300
278
U/kg) which is not the case in standard bakery products.
279 280
Analysis of MTG-treated bread for gliadin deamidation
281
Antibodies raised against deamidated α2-gliadin 33mer epitopes, either specific for
282
the deamidated epitope (A057) or recognizing both unmodified and deamidated
283
33mer (A062) were used to detect possible deamidation of gluten by MTG.13
284
Western blot analysis showed that in the industrially prepared bread samples (0, 2, 4
285
and 8 U MTG/kg flour), basically no staining was observed using deamidation-
286
specific antibody A057 (Figure 4). However, for the 2,000 U MTG/kg controls,
287
staining of multiple bands with a molecular size of 36-40 kDa was observed in every
288
extract. Also the positive control DGPx2 was intensively stained. A062 finally
289
showed, an increase in staining intensity of bands with a molecular size of approx. 37
290
kDa in the albumin/globulin and gliadin extracts for samples with 4 U and 8 U
291
MTG/kg flour. As for the more sensitive antibody A057 (Figure 1) no signal increase
292
was observed, the growing intensity with A062 (the antibody shows some selectivity
293
for the unmodified gliadin, Figure 1) should be due to unmodified gliadin. It can be
294
speculated that MTG-treatment increases accessibility of the (unmodified) epitope.
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At least using monoclonal antibody A057 specific to deamidated α2-gliadin 33mer
296
neither increase nor presence of any deamidated gliadin could be detected. To
297
further examine this finding, celiac disease patients’ sera were used in Western blot
298
analysis of the bread extracts.
299 300
Analysis of MTG-treated bread using celiac disease patients’ sera
301
In total, 26 celiac disease patients’ sera were characterized for their antibody-titers
302
against unmodified gliadin and deamidated gliadin peptide (DGP). All sera were
303
positive for DGP antibodies (either IgA or IgG) with the exception of two sera (225224
304
and 232559), which were in addition negative for both types of anti-gliadin antibodies.
305
Further, half of the sera were negative for anti-gliadin antibodies (IgA and IgG), 5 of
306
them with a high anti-DGP-titer. The sera were grouped in high (>50 U/mL, Table 1)
307
and low titer anti-DGP (500 U/kg. While we were able to generate deamidated gliadin peptides in
338
high-dose MTG-treated bread (2,000 U/kg) no deamidated gliadin could be detected
339
in bread produced with standard bakery amounts of MTG. In addition, MTG has a
340
more than 10-fold transamidation preference. In the presence of sufficient amounts of
341
primary amine substrates deamidation can be blocked completely.10 Taking these
342
observations together, deamidation probably may take place only at high dose MTG 15 ACS Paragon Plus Environment
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when primary amines are not available in sufficient amounts anymore or already
344
depleted by the MTG-reaction. This is supported by the finding from Mazzeo et al.,
345
who did not detect deamidated gliadin by nano-HPLC-ESI-MS/MS upon addition of
346
even 8,000 U MTG/kg flour in the presence of 20 mM Lysine-ethylester.45
347
To our knowledge so far one example for a food based trigger for celiac disease is
348
described. In the 1980s the Swedish national recommendation to postpone
349
introduction of gluten to 6 months old children resulted in an immediate increase of
350
celiac disease in Sweden. Within 3 years celiac disease incidence almost
351
quadrupled.46 MTG has been introduced in bakery products in the mid-1990s.
352
However, no evidence for an immediate increase in celiac disease incidence at the
353
same time has been reported. Thus, epidemiological data do not support a celiac
354
disease triggering impact by MTG.
355
It is a fact that MTG is able to deamidate gliadin rendering the peptides immunotoxic,
356
as shown by ourselves and other groups. Therefore, it cannot be excluded that MTG
357
treatment of bread has an impact on celiac disease. However, in the light of our
358
results and the published data mentioned above, citing Philippus Theophrastus
359
Aureolus Bombastus von Hohenheim (1493 – 1541) - also known as Paracelsus -
360
seems appropriate: “sola dosis facit venenum – the dose makes the poison”.
361
In summary, standard bakery doses of MTG are magnitudes lower than used in
362
academic examinations. They do not lead to detectable amounts of deamidated
363
gliadin using a deamidated gliadin specific monoclonal antibody and celiac disease
364
patients’ sera in western blotting. Especially no increase in deamidated gliadin was
365
found.
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Abbreviations used:
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CD, celiac disease; DGP, deamidated gliadin peptide; ELISA, enzyme linked immune
370
sorbent assay; MTG, microbial transglutaminase; DGP, deamidated gliadin peptide;
371
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; DTE,
372
dithioerythritol.
373 374 375
Acknowledgement:
376
Preparation of MTG-containing bread by Norman Burkhardt (AB Enzymes) and
377
preparation of graphics by Katrin Bott-Fischer (Zedira) is gratefully acknowledged.
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References
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(1)
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Figure captions
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Figure 1. Specificity of monoclonal antibodies used for the analysis of MTG-treated
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bread. Recombinant proteins carrying different unmodified and deamidated
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immunotoxic peptide sequences isolated from wheat gliadin were analyzed using dot
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blot to identify specific binding patterns of the antibodies.
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Figure 2. SDS-PAGE and Western blot analysis of wheat protein fractions extracted
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from bread prepared without (0), 2, 4 and 8 U MTG/kg flour. A, B, C: silver-stained
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gels; D, E, F: Western blots using monoclonal antibody A011 specific to gliadin as
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detection antibody. Gliadin extract from 2,000 U MTG/kg flour and DGPx2 were
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deamidated gliadin positive controls.
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Figure 3. Western blot analysis of MTG treated bread extracts using antibodies
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against unmodified gliadin. Protein extracts as described in figure 2. A, B, C: Western
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blots with gliadin specific antibody A054; D, E, F: Western blots with gliadin specific
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antibody A064. DGPx2: deamidated gliadin control. 26mer GP: non-deamidated
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gliadin control (the multiple bands visible in the blots are essentially due to multimers
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or degradation products of recombinant 26mer GP visible at high concentrations in
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combination with sensitive detection).
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Figure 4. Western blot analysis of extracts from MTG treated bread samples using
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antibodies against deamidated gliadin. Protein extracts as described in figure 2. A, B, 24 ACS Paragon Plus Environment
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C: Western blots with gliadin specific antibody A057; D, E, F: Western blots with
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gliadin specific antibody A062. DGPx2: deamidated gliadin control.
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Figure 5. Western blot analysis of MTG treated bread samples using high titer celiac
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disease patients’ sera. Protein extracts as described in figure 2. A, B, C: Western
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blots with high titer sera pool of IgG type; D, E, F: Western blots with high titer sera
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pool of IgA type. DGPx2: deamidated gliadin control. 26mer GP: non-deamidated
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gliadin control.
535 536
Figure 6. Western blot analysis of MTG treated bread samples using low titer celiac
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disease patients’ sera. Protein extracts as described in figure 2. A, B, C: Western
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blots with low titer sera pool of IgG type; D, E, F: Western blots with low titer sera
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pool of IgA type. DGPx2: deamidated gliadin control. 26mer GP: non-deamidated
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gliadin control.
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Tables Table 1: Celiac disease patients’ sera with high titers of anti-DGP and anti-gliadin antibodies (IgA and IgG; c/o = cut off).
Serum No. 221096 223261 224184 224187 224234 225254 225380 225833 225872 227542 232554 240820 240993 232394
High titer sera, either IgA or IgG > 50 U/mL (n = 14) DGPx1 IgA DGPx1 IgG Gliadin IgA c/o = 8 c/o = 7 c/o = 14 31 65 8 100 33 >100 96 99 68 6 50 20 70 12 >100 23 >100 4 100 3 41 >100 24 100 0 >100 >100 40 27 >100 11 >100 87 >100 >100 >100 55 27 60 16
Gliadin IgG c/o = 14 7 4 9 3 3 80 16 >100 8 14 2 12 10 2
Table 2: Celiac disease patients’ sera with low titers of anti-DGP and anti-gliadin antibodies (IgA and IgG; c/o = cut off), respectively.
Serum No. 221057 223253 223260 223333 224360 225224 225292 225387 232553 232559 225938 223332
Low titer sera, IgA and IgG < 50 U/mL (n = 12) DGPx1 IgA DGPx1 IgG Gliadin IgA c/o = 8 c/o = 7 c/o = 14 33