1. Enzyme-Linked Immunoassay Tests for Gluten Content in Food

2. Celiac Disease
Nearly 3 million United States citizens suffer from celiac disease
Permanent intolerance to gluten
Small intestine damaged

3. Symptoms
Bloating
Cramping
Intestinal gas
Diarrhea
Constipation

4. Treatment for Celiac Disease
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

5. 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)

6. What Is Gluten?
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

7. 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)

8. Gluten Testing Performed in food science laboratories
Food scientists, other specialists

9. Advantages and Disadvantages of Each Method
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)
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

10. 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

11. 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)

12. Terms to Understand 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

13. 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
Step 2
An antigen (toxin that contains unknown quantity of gluten) is added to antibody A coated microwell

14. Sandwich ELISA Protocol (continued)
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

15. Sandwich ELISA Protocol (continued)
Step 4 (continued)
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

16. 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

17. Sandwich ELISA Protocol (continued)
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

18. How Each Gluten Method Works

19. A Second Diagram of How Each Method Works
Sandwich ω-ELISA Method
1. Antibody coated 2. Gliadin (􀁣) in 3. HRP enzyme-labelled 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.

20. How Second Method Works
Competitive ELISA Method

21. How Competitive R5 ELISA Works
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)
Step 2
Unlabeled samples including unknowns and standards (knowns) are added to microwells and incubated until equilibrium (binding site potential has reached its maximum)

22. How Competitive R5 Elisa Works
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

23. 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

24. How Competitive R5 ELISA Works
Step 6
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