Enzyme-Linked Immunoassay Tests
for Gluten Content in Food
Celiac Disease
Nearly 3 million United States citizens
suffer from celiac disease
 Permanent intolerance to gluten
 Small intestine damaged

Symptoms
Bloating
 Cramping
 Intestinal gas
 Diarrhea
 Constipation
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Treatment for Celiac Disease
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Gluten Free Diet
◦ Avoid rye, wheat, barley, related cereal
grains
◦ May consume rice, buckwheat, corn, quinoa
◦ Oats contain traces gluten that can cause
mild symptoms
Gluten Free Food Labeling

Federal Drug Administration (FDA)
◦ Food Allergen Labeling and Consumer Protection
Act of 2004
◦ Final rule by 2008 still pending
◦ No gluten containing ingredients: rye, wheat,
barley, crossbred
◦ Food must be processed to remove gluten
◦ Final food product <20 parts per million (ppm)
What Is Gluten?
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Complexed water-insoluble proteins: gliadin
and glutenin
Found in rye, wheat and barley seeds
Allows dough to bind, gives elasticity,
rubberiness
Spongy consistency to breads, cakes,
other baked products
Two Gluten Analysis Methods
Available To Food Manufacturers

Method One: sandwich w-gliadin enzyme
immunoabsorbent assay (ELISA)
◦ Officially approved in 1991 by Association of
Analytical Communities (AoAC)
◦ ω means omega (protein in gliadin)
•
Method Two: competitive sandwich R5
immunoabsorbent assay (R5 ELISA)
◦ Officially approved in 2006 by Codex
Alimentarius Commission
◦ Joint body of World Health Organization (WHO)
Gluten Testing

Performed in food science laboratories
◦ Food scientists, other specialists
Sandwich ELISA
R5 ELISA
Developed by Skerritt and Hill
 Requires 2 epitopes (antibody
binding sites)
 Underestimates barley
protein content from
hydrolyzed and partially
hydrolyzed proteins (proteins
broken apart)
 Can detect both heated
(denatured) and unheated
proteins at gluten levels >/150
ppm (150 milligrams per
kilogram)
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Developed by Mendez
Requires one specific binding site
(R5 monoclonal antibody)
Accurately detects highly toxic
heat resistant protein
Overestimates barley protein
content
Unable to measure hydrolyzed
gluten proteins
Can detect both heated and
unheated proteins
Recognizes all wheat, barley and
rye gluten at detection level </3
ppm and able to measure </5 ppm
Advantages and Disadvantages
of Each Method
Terms to Understand
Antibody- protein produced by immune system in
response to presence of a foreign substance
(antigen). Its main function is to neutralize it
(make harmless)
 Antigen- protein, toxin or other foreign substance
that causes body to react by producing antibodies
(antigen is the reagent)
 Enzyme- proteins that make reactions occur
 Epitope- structural site on an antigen (invader)
specific to only one particular antibody

Terms to Understand
Enzyme linked antigen-an enzyme attached to an
antigen to be used as a marker to detect a target
protein (in this case a gluten rich protein)
 Horseradish peroxidase (HRP)- an enzyme used
to label antigens and their antibodies
 Microtiter plate- a standard size plate that
typically contains 96 small test tube (HRP)
containers (wells)
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Terms to Understand
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Microwell- test tube container
Parts per million- milligrams per kilogram (ratio =
one to one million)
Substrate- a substance or material on which an
enzyme attaches and reacts to make a change occur
Solid phase support - microtitre plate wells are
example in ELISA
Spectrophotometer- instrument that measures
amount of ultraviolet light absorbed by a substance
Protocol for Sandwich ELISA

Step 1
◦ A plastic test tube (microwell) that contains
solid phase support on its base is coated well
with purified antibody A
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Step 2
◦ An antigen (toxin that contains unknown
quantity of gluten) is added to antibody A
coated microwell
Sandwich ELISA Protocol
(continued)
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Step 3
◦ Unbound products are removed with a mild detergent
Step 4
◦ Antibody detection joined to enzyme (horseradish
peroxidase commonly used to detect antigen: gliadin in
gluten detection)
◦ Antibody B recognizes second separate binding site
(epitope) on antigen (gliadin bound to antibody A) and
binds “sandwich” complex is formed
Sandwich ELISA Protocol
(continued)
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Step 4 (continued)
o Antibody B is used to increase likelihood that
particular gluten is present
Step 5
◦ Colorimetric substrate (substrate that measures
gluten content by amount of color intensity) is
added to measure the amount of gluten that is
detected by labeled second antibody B and its
joined enzyme
Sandwich ELISA Protocol
(continued)

Step 6
◦ Spectrophotometer or ELISA microplate
reader
◦ Both instruments measure intensity of
ultraviolet light absorbed by substance
◦ Microplate reader reads entire microtiter
plate at one time
Sandwich ELISA Protocol
(continued)
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Step 7
◦ Spectrophotometer reads individual microwells
◦ Adjusting spectrophotometer or microplate reader
◦ Adjust to specific optical density such as 450 optical
density
◦ Record results for each sample
◦ Graph results
 Y axis (dependent variable): optical density
 X axis (independent variable): of concentration of antigen
present
 Plot points on graph
 Create linear curve
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How Each Gluten Method Works
A Second Diagram of How Each
Method Works
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Sandwich ω-ELISA Method
1. Antibody coated 2. Gliadin (􀁣) in 3. HRP enzymelabelled 2nd 4. Addition of TMB substrate,
plastic micro-well. ethanolic food antibody binds in
turn which develops blue
extract binds to to gliadin bound to colour in
presence of
antibody on well. antibody on well. HRP enzyme.
How Second Method Works
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Competitive ELISA Method
How Competitive R5 ELISA Works
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Step 1:
◦ Unlabeled (no enzyme attached) purified primary
antibody A is coated onto separate small test
tubes (microwells) of a microtiter plate (usually
contains 96 wells for ELISA gluten testing
method)
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Step 2
◦ Unlabeled samples including unknowns and
standards (knowns) are added to microwells and
incubated until equilibrium (binding site potential
has reached its maximum)
How Competitive R5 Elisa Works
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Step 3
◦ To antibody A, standard, unknown complex in
microwell, unlabeled antigen conjugated (attached) to
detection enzyme (Strept Avidin Horseradish
Peroxidase is added
◦ antigen joined to enzyme will bind to
primary antibody A at its unoccupied
binding sites
the more antigen in unknown and
standard (known) the lower number of
binding sites available to antigen conjugated
to enzyme
How Competitive R5 ELISA Works
Step 4
Substrate added and incubated
enzyme reacts with substrate to
release blue color
intensity of blue color determines
antigen gluten content
 Step 5
Acid stop solution added
stops reaction
changes solution from blue to yellow
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How Competitive R5 ELISA Works
Step 6
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Use spectrophotometer or microplate
reader to determine gluten content by
measuring color intensity
• Step 7
•
Graph results
Y axis (dependent variable): optical density
X axis (independent variable): concentration
antigen present (nanograms)
Plot points on graph
Create linear curve
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