Gluten determination from foods

Introduction:

Gluten (from Latin gluten, "glue") is a group of proteins, called prolamins and glutelins, which occur

with starch in the endosperm of various cereal grains. 
It is found in related wheat species and hybrids, (such as spelt, khorasan, emmer, einkorn,
and triticale), barley, rye, and oats, as well as products derived from these grains, such
as breads and malts.

Chemical structure:
Gluten is a mixture of prolamin proteins (in wheat are called gliadins) and glutelines ( in wheat are
called glutenines).
Based on their solubility in alcoholic solutions, these subunits are divided as follows:
 Prolamines are the solubile compounds in alcoholic solutions;
 Glutenines are the insolubile compounds in alcoholic solutions;
Gluten proteins appear to have a significant amount of glutamine and proline amino acid residues present
in their primary structures.
A. Prolamins (gliadins) are represented as single chain polypeptides, and it is accepted that
gliadins are divided into four major groups (from fastest mobility to slowest): α-, β-, γ-, and ω-
gliadins, according to their electrophoretic mobility in SDS-PAGE at low pH . Precisely there is:
o ω- gliadin;
o α-gliadin;
o β- gliadin;
o γ-gliadins.
There is also a difference in the number and properties of prolamin polypeptides. Despite these
variations, all prolamins are related and, usually referred to as three broad groups:
-sulphur-rich (S-rich);
-sulphur-poor (S-poor);
- high molecular weight (HMW) prolamins.  (Fig.1)

Figura 1. Classification of prolamin superfamily

 The proteins and polypeptides within these groups possess similar structures:
o signal peptide for translocation into cellular compartments;
o a non-repetitive N-terminal region;
o a non-repetitive C-terminal region;
o a long repetitive central region.(Fig.2)

Figura 2. The structure of prolamines

B. Glutenins consist of subunits and are usually divided into two classes according to their
molecular weight defined by SDS-PAGE: high molecular weight glutenin subunits (HMW-GS)
and low molecular weight glutenin subunits (LMW-GS).

Gluten Intolerance pathogenesis

Primarily, gluten is a source of flour and, consequently, bread and flour products. Consumption of
gluten-containing food makes such food an immune system target. Digested gluten is a reason for the
emergence of different antigens and immunogens. Gluten is impregnable by the gastric, pancreatic and
intestinal digestive proteases of people carrying HLA-DQ2 or/and DQ8 haplotype. HLA-DQ is a part of
the MHC class II antigen-presenting receptor system and distinguishes its own and foreign cells.
Gluten peptides involved in celiac disease, are divided into two groups:
- toxic peptides;
- immunogenic peptides.
Toxic peptides are capable of inducing mucosal damage when administered in vivo on the intestine,
whereas peptide is considered to be immunogenic if it is able to specifically stimulate HLA-DQ-restricted
T cell lines isolated from peripheral blood of CD patients.
Peptides differ from each other in the degree of the immunogenicity. It is remarkable that some are
immunodominant, meaning that they evoke strong T cell response in almost every CD patient, whereas
the immunogenic do not.
 All gluten proteins (gliadin and glutenin from wheat, hordein from barley, secalin from rye and avenin
from oat) possess their own sets of toxic and immunogenic peptides (or epitopes) with distinct
immunogenicities. However, gliadin peptides are known to be the most toxic and numerous, specifically
derived from α- and γ-gliadin: the strongest and most common adaptive response to gluten is directed
toward an α2-gliadin fragment of 33 amino acids in length.
Digestion of gliadin results in the emergence of two pieces: 25-mer (p31-55, it can be degraded to
smaller peptides) and 33-mer (p57-89).
The α-gliadin 33-mer is one of the digestion-resistant gluten peptides that is highly reactive to
isolated celiac T cells and is the main immunodominant toxic peptide in celiac patients. It is located in
the N-terminal repetitive region of α-gliadin and contains six overlapping copies of three different DQ2-
restricted epitopes (Fig 3).

Figura 3. The 33-mer fragment from the structure of alpha-gliadine.

Gluten intolerance
The term gluten intolerance may refer to three types of human disorders: autoimmune celiac disease
(CD), allergy to wheat and non-celiac gluten sensitivity (NCGS).

 Wheat Allergy

Wheat flour triggers IgE-mediated food allergy and is one of the top eight food allergens. Wheat allergy
commonly develops in childhood. When an allergen specifically binds to IgE antibodies, it induces the
activation of mast cells and basophils. Wheat allergy symptoms include: swelling, itching or irritation of
the mouth or throat, hives, itchy rash or swelling of the skin, nasal congestion, headache, difficulty
breathing, cramps, nausea or vomiting, diarrhea and anaphylaxis.

 Celiac disease

Celiac disease is a serious autoimmune disease that occurs in genetically predisposed people where the
ingestion of gluten leads to damage in the small intestine. It is estimated to affect 1 in 100 people
worldwide. Digestive symptoms are more common in infants and children. Here are the most common
symptoms found in children: abdominal bloating and pain, chronic diarrhea, vomiting, constipation, pale,
foul-smelling, or fatty stool, iron-deficiency anemia and weight loss. Adults are less likely to have
digestive symptoms, with only one-third experiencing diarrhea. Adults are more likely to have:
unexplained iron-deficiency anemia, fatigue, bone or joint pain, arthritis, osteoporosis or osteopenia (bone
loss), liver and biliary tract disorders (transaminitis, fatty liver, primary sclerosing cholangitis, etc.).
 A simple blood test is available to test for celiac disease. People with celiac disease who eat gluten have
higher than normal levels of certain antibodies in their blood. These antibodies are produced by the
immune system because it views gluten (the proteins found in wheat, rye and barley) as a threat. For most
children and adults, the best way to test for celiac disease is with the Tissue Transglutaminase IgA
antibody, plus an IgA antibody in order to ensure that the patient generates enough of this antibody to
render the celiac disease test accurate. Celiac disease is a chronic autoimmune disease, which means that
you cannot “grow out” of it. The treatment for both celiac disease and non-celiac gluten/wheat sensitivity
is lifelong adherence to a strict gluten-free diet. Only food and beverage with a gluten content less than 20
parts per million (ppm) is allowed. The gluten-free diet heals the villous atrophy in the small intestine,
causing symptoms to resolve. Following the gluten-free diet also helps prevent future complications,
including malignancies.

 Gluten intolerance ( non-celiac gluten intolerance-NCGS)

The first reported NCGS cases were described as longstanding and previously unresolved history of
abdominal pain, discomfort, bloating, altered bowel habit and fatigue with exclusion of celiac disease.
NCGS is more frequently diagnosed in adults rather than in children. In most cases, NCGS reveals itself a
few hours after gluten digestion. Similarly to CD patients, patients with NCGS suffer from nutritional
deficiencies, coexisting autoimmunity, and a decreased bone mineral density compared with the general
population. In NCGS there is no anti-tTG2 antibodies identified. Gluten only triggered an innate immune
response in NCGS and provoked an additional adaptive immune response with increased expression of
IL-6, IL-21, IL-17 and IFN-γ. However, gastrointestinal symptoms other than intestinal permeability and
adaptive immune responses are not involved in the process. In NCGS, gliadins do not induce mucosal
inflammation in vitro or the activation of basophils as seen in CD( celiac disease).

Practical Application

Determination of gluten in foods using AgraStrip® Gluten 1 rapid tests

Principle

The determination principle is based on the gluten agglutination reaction on the surface of the reactive
band with the appearance of a color change (red). The examination is performed with the aid of reactive
strips that qualitatively determine the presence of gluten( from 5ppm) in the tested food product. This test
is based on a method of immunometric determination (sandwich type). Both the anti-gliadin monoclonal
antibody as well as anti-gliadin antibody labeled with an enzyme that will cause a colour change are fixed
on the strip surface. The antigen gliadin will react simultaneously with the 2 antibodies and then the color
reaction will occur. The changes will be compared with the standard scale delivered with the test (Fig.4).
Fig 4. Antiboby G12 binds to the 33-mer fragment on the alpha-gliadin
Close extraction tube with
Homogenize the sample and
cap and shake vigorously by
weigh 0.2 g of sample and
hand for 1 minute. Remove
add to the extraction tube Fill
extraction tube cap and
tube with extraction solution
replace with dropper tip
to level shown in picture.
Close dilution tube with Take off cap and place the
cap and shake vigorously strip vertically in dilution
by hand for 15 seconds. tube and allow liquid to wick
to level shown in the
diagram (this takes about 45
seconds).
Transfer 3 drops from
extraction tube to dilution tube
and fi ll up with dilution buffer
to desired cutoff level indicated
by 5, 10 and 20 ppm marks on
dilution tube.
Remove the strip from the
dilution tube and place it
upright into slot of the tube
holder provided. Allow the
strip to develop for 10 minutes
and then read off the result
immediately.
Reagents and Materials
 Reactive strip
 Extraction solution
 Dilution solution
 Vial for extraction procedure
 Vial for dilution
 Analyzed material

Procedure

1. Homogenize the sample and weigh 0.2 g of sample and add to the extraction tube Fill tube with
extraction solution to level shown in picture.
2. Close extraction tube with cap and shake vigorously by hand for 1 minute. Remove extraction
tube cap and replace with dropper tip
3. Transfer 3 drops from extraction tube to dilution tube and fi ll up with dilution buffer to desired
cutoff level indicated by 5, 10 and 20 ppm marks on dilution tube.
4. Close dilution tube with cap and shake vigorously by hand for 15 seconds.
5. Take off cap and place the strip vertically in dilution tube and allow liquid to wick to level shown
in the diagram (this takes about 45 seconds).
6. Remove the strip from the dilution tube and place it upright into slot of the tube holder provided.
Allow the strip to develop for 10 minutes and then read off the result immediately.

Results: