Enzyme-Linked Immunoassay Tests for Gluten Content in Food

Celiac DiseaseNearly 3 million United States

citizens suffer from celiac diseasePermanent intolerance to glutenSmall intestine damaged

Symptoms

Bloating CrampingIntestinal gasDiarrheaConstipation

Treatment for Celiac DiseaseGluten 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 LabelingFederal Drug Administration

(FDA)Food Allergen Labeling and Consumer

Protection Act of 2004Final rule by 2008 still pendingNo gluten containing ingredients rye

wheat barley crossbredFood must be processed to remove

glutenFinal food product lt20 parts per million

(ppm)

What Is GlutenComplexed water-insoluble

proteins gliadin and gluteninFound in rye wheat and barley

seedsAllows dough to bind gives

elasticity rubberinessSpongy consistency to breads

cakes other baked products

Two Gluten Analysis Methods Available To Food ManufacturersMethod One sandwich w-gliadin

enzyme immunoabsorbent assay (ELISA)Officially approved in 1991 by Association of

Analytical Communities (AoAC) ω means omega (protein in gliadin)

bull 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 TestingPerformed in food science

laboratoriesFood scientists other specialists

Advantages and Disadvantagesof 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 gt150 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 lt3 ppm and able to measure lt5 ppm

Terms to UnderstandAntibody- 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 occurEpitope- structural site on an antigen

(invader) specific to only one particular antibody

Terms to UnderstandEnzyme 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)

Terms to UnderstandMicrowell- 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 ELISAStep 1

A plastic test tube (microwell) that contains solid phase support on its base is coated well with purified antibody A

Step 2An antigen (toxin that contains

unknown quantity of gluten) is added to antibody A coated microwell

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

Celiac DiseaseNearly 3 million United States

citizens suffer from celiac diseasePermanent intolerance to glutenSmall intestine damaged

Symptoms

Bloating CrampingIntestinal gasDiarrheaConstipation

Treatment for Celiac DiseaseGluten 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 LabelingFederal Drug Administration

(FDA)Food Allergen Labeling and Consumer

Protection Act of 2004Final rule by 2008 still pendingNo gluten containing ingredients rye

wheat barley crossbredFood must be processed to remove

glutenFinal food product lt20 parts per million

(ppm)

What Is GlutenComplexed water-insoluble

proteins gliadin and gluteninFound in rye wheat and barley

seedsAllows dough to bind gives

elasticity rubberinessSpongy consistency to breads

cakes other baked products

Two Gluten Analysis Methods Available To Food ManufacturersMethod One sandwich w-gliadin

enzyme immunoabsorbent assay (ELISA)Officially approved in 1991 by Association of

Analytical Communities (AoAC) ω means omega (protein in gliadin)

bull 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 TestingPerformed in food science

laboratoriesFood scientists other specialists

Advantages and Disadvantagesof 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 gt150 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 lt3 ppm and able to measure lt5 ppm

Terms to UnderstandAntibody- 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 occurEpitope- structural site on an antigen

(invader) specific to only one particular antibody

Terms to UnderstandEnzyme 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)

Terms to UnderstandMicrowell- 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 ELISAStep 1

A plastic test tube (microwell) that contains solid phase support on its base is coated well with purified antibody A

Step 2An antigen (toxin that contains

unknown quantity of gluten) is added to antibody A coated microwell

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

Symptoms

Bloating CrampingIntestinal gasDiarrheaConstipation

Treatment for Celiac DiseaseGluten 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 LabelingFederal Drug Administration

(FDA)Food Allergen Labeling and Consumer

Protection Act of 2004Final rule by 2008 still pendingNo gluten containing ingredients rye

wheat barley crossbredFood must be processed to remove

glutenFinal food product lt20 parts per million

(ppm)

What Is GlutenComplexed water-insoluble

proteins gliadin and gluteninFound in rye wheat and barley

seedsAllows dough to bind gives

elasticity rubberinessSpongy consistency to breads

cakes other baked products

Two Gluten Analysis Methods Available To Food ManufacturersMethod One sandwich w-gliadin

enzyme immunoabsorbent assay (ELISA)Officially approved in 1991 by Association of

Analytical Communities (AoAC) ω means omega (protein in gliadin)

bull 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 TestingPerformed in food science

laboratoriesFood scientists other specialists

Advantages and Disadvantagesof 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 gt150 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 lt3 ppm and able to measure lt5 ppm

Terms to UnderstandAntibody- 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 occurEpitope- structural site on an antigen

(invader) specific to only one particular antibody

Terms to UnderstandEnzyme 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)

Terms to UnderstandMicrowell- 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 ELISAStep 1

A plastic test tube (microwell) that contains solid phase support on its base is coated well with purified antibody A

Step 2An antigen (toxin that contains

unknown quantity of gluten) is added to antibody A coated microwell

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

Treatment for Celiac DiseaseGluten 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 LabelingFederal Drug Administration

(FDA)Food Allergen Labeling and Consumer

Protection Act of 2004Final rule by 2008 still pendingNo gluten containing ingredients rye

wheat barley crossbredFood must be processed to remove

glutenFinal food product lt20 parts per million

(ppm)

What Is GlutenComplexed water-insoluble

proteins gliadin and gluteninFound in rye wheat and barley

seedsAllows dough to bind gives

elasticity rubberinessSpongy consistency to breads

cakes other baked products

Two Gluten Analysis Methods Available To Food ManufacturersMethod One sandwich w-gliadin

enzyme immunoabsorbent assay (ELISA)Officially approved in 1991 by Association of

Analytical Communities (AoAC) ω means omega (protein in gliadin)

bull 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 TestingPerformed in food science

laboratoriesFood scientists other specialists

Advantages and Disadvantagesof 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 gt150 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 lt3 ppm and able to measure lt5 ppm

Terms to UnderstandAntibody- 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 occurEpitope- structural site on an antigen

(invader) specific to only one particular antibody

Terms to UnderstandEnzyme 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)

Terms to UnderstandMicrowell- 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 ELISAStep 1

A plastic test tube (microwell) that contains solid phase support on its base is coated well with purified antibody A

Step 2An antigen (toxin that contains

unknown quantity of gluten) is added to antibody A coated microwell

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

Gluten Free Food LabelingFederal Drug Administration

(FDA)Food Allergen Labeling and Consumer

Protection Act of 2004Final rule by 2008 still pendingNo gluten containing ingredients rye

wheat barley crossbredFood must be processed to remove

glutenFinal food product lt20 parts per million

(ppm)

What Is GlutenComplexed water-insoluble

proteins gliadin and gluteninFound in rye wheat and barley

seedsAllows dough to bind gives

elasticity rubberinessSpongy consistency to breads

cakes other baked products

Two Gluten Analysis Methods Available To Food ManufacturersMethod One sandwich w-gliadin

enzyme immunoabsorbent assay (ELISA)Officially approved in 1991 by Association of

Analytical Communities (AoAC) ω means omega (protein in gliadin)

bull 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 TestingPerformed in food science

laboratoriesFood scientists other specialists

Advantages and Disadvantagesof 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 gt150 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 lt3 ppm and able to measure lt5 ppm

Terms to UnderstandAntibody- 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 occurEpitope- structural site on an antigen

(invader) specific to only one particular antibody

Terms to UnderstandEnzyme 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)

Terms to UnderstandMicrowell- 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 ELISAStep 1

A plastic test tube (microwell) that contains solid phase support on its base is coated well with purified antibody A

Step 2An antigen (toxin that contains

unknown quantity of gluten) is added to antibody A coated microwell

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

What Is GlutenComplexed water-insoluble

proteins gliadin and gluteninFound in rye wheat and barley

seedsAllows dough to bind gives

elasticity rubberinessSpongy consistency to breads

cakes other baked products

Two Gluten Analysis Methods Available To Food ManufacturersMethod One sandwich w-gliadin

enzyme immunoabsorbent assay (ELISA)Officially approved in 1991 by Association of

Analytical Communities (AoAC) ω means omega (protein in gliadin)

bull 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 TestingPerformed in food science

laboratoriesFood scientists other specialists

Advantages and Disadvantagesof 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 gt150 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 lt3 ppm and able to measure lt5 ppm

Terms to UnderstandAntibody- 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 occurEpitope- structural site on an antigen

(invader) specific to only one particular antibody

Terms to UnderstandEnzyme 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)

Terms to UnderstandMicrowell- 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 ELISAStep 1

A plastic test tube (microwell) that contains solid phase support on its base is coated well with purified antibody A

Step 2An antigen (toxin that contains

unknown quantity of gluten) is added to antibody A coated microwell

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

Two Gluten Analysis Methods Available To Food ManufacturersMethod One sandwich w-gliadin

enzyme immunoabsorbent assay (ELISA)Officially approved in 1991 by Association of

Analytical Communities (AoAC) ω means omega (protein in gliadin)

bull 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 TestingPerformed in food science

laboratoriesFood scientists other specialists

Advantages and Disadvantagesof 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 gt150 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 lt3 ppm and able to measure lt5 ppm

Terms to UnderstandAntibody- 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 occurEpitope- structural site on an antigen

(invader) specific to only one particular antibody

Terms to UnderstandEnzyme 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)

Terms to UnderstandMicrowell- 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 ELISAStep 1

A plastic test tube (microwell) that contains solid phase support on its base is coated well with purified antibody A

Step 2An antigen (toxin that contains

unknown quantity of gluten) is added to antibody A coated microwell

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

Gluten TestingPerformed in food science

laboratoriesFood scientists other specialists

Advantages and Disadvantagesof 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 gt150 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 lt3 ppm and able to measure lt5 ppm

Terms to UnderstandAntibody- 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 occurEpitope- structural site on an antigen

(invader) specific to only one particular antibody

Terms to UnderstandEnzyme 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)

Terms to UnderstandMicrowell- 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 ELISAStep 1

A plastic test tube (microwell) that contains solid phase support on its base is coated well with purified antibody A

Step 2An antigen (toxin that contains

unknown quantity of gluten) is added to antibody A coated microwell

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

Advantages and Disadvantagesof 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 gt150 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 lt3 ppm and able to measure lt5 ppm

Terms to UnderstandAntibody- 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 occurEpitope- structural site on an antigen

(invader) specific to only one particular antibody

Terms to UnderstandEnzyme 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)

Terms to UnderstandMicrowell- 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 ELISAStep 1

A plastic test tube (microwell) that contains solid phase support on its base is coated well with purified antibody A

Step 2An antigen (toxin that contains

unknown quantity of gluten) is added to antibody A coated microwell

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

Terms to UnderstandAntibody- 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 occurEpitope- structural site on an antigen

(invader) specific to only one particular antibody

Terms to UnderstandEnzyme 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)

Terms to UnderstandMicrowell- 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 ELISAStep 1

A plastic test tube (microwell) that contains solid phase support on its base is coated well with purified antibody A

Step 2An antigen (toxin that contains

unknown quantity of gluten) is added to antibody A coated microwell

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

Terms to UnderstandEnzyme 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)

Terms to UnderstandMicrowell- 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 ELISAStep 1

A plastic test tube (microwell) that contains solid phase support on its base is coated well with purified antibody A

Step 2An antigen (toxin that contains

unknown quantity of gluten) is added to antibody A coated microwell

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

Terms to UnderstandMicrowell- 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 ELISAStep 1

A plastic test tube (microwell) that contains solid phase support on its base is coated well with purified antibody A

Step 2An antigen (toxin that contains

unknown quantity of gluten) is added to antibody A coated microwell

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

Protocol for Sandwich ELISAStep 1

A plastic test tube (microwell) that contains solid phase support on its base is coated well with purified antibody A

Step 2An antigen (toxin that contains

unknown quantity of gluten) is added to antibody A coated microwell

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

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 ldquosandwichrdquo complex is formed

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

Sandwich ELISA Protocol (continued)Step 4 (continued)oAntibody B is used to increase

likelihood that particular gluten is present

Step 5Colorimetric 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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

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)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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

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

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

How Each Gluten Method Works

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

A Second Diagram of How Each Method Works

Sandwich ω-ELISA Method

1 Antibody coated 2 Gliadin (1048675) 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

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

How Second Method WorksCompetitive ELISA Method

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

How Competitive R5 ELISA WorksStep 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 2Unlabeled 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

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 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

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

How Competitive R5 ELISA Works Step 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

How Competitive R5 ELISA Works Step 4o Substrate added and incubatedbull enzyme reacts with substrate to release blue color intensity of blue color determines antigen gluten contentStep 5 Acid stop solution added stops reaction changes solution from blue to yellow

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)

How Competitive R5 ELISA Works Step 6bull Use spectrophotometer or microplate reader to determine gluten content by measuring color intensitybull Step 7bull Graph results Y axis (dependent variable) optical density X axis (independent variable) concentration antigen present (nanograms) Plot points on graph Create linear curve

• Enzyme-Linked Immunoassay Tests for Gluten Content in Food
• Celiac Disease
• Symptoms
• Treatment for Celiac Disease
• Gluten Free Food Labeling
• What Is Gluten
• Two Gluten Analysis Methods Available To Food Manufacturers
• Gluten Testing
• Advantages and Disadvantages of Each Method
• Terms to Understand
• Terms to Understand (2)
• Terms to Understand (3)
• Protocol for Sandwich ELISA
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (2)
• Sandwich ELISA Protocol (continued)
• Sandwich ELISA Protocol (continued) (3)
• How Each Gluten Method Works
• A Second Diagram of How Each Method Works
• How Second Method Works
• How Competitive R5 ELISA Works
• How Competitive R5 Elisa Works
• How Competitive R5 ELISA Works
• How Competitive R5 ELISA Works (2)