ELISA: Enzyme-Linked Immunosorbent Assay

ELISA: Enzyme-Linked Immunosorbent Assay

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ELISA detects substances with antigenic properties (mainly proteins)

Based on enzymatic color-reaction

Basic principle of ELISA

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Enzyme is used to detect the binding of Antibody - Antigen

Enzyme converts colorless substrate into colored product, indicating the presence of Antibody - Antigen complex

ELISA can be used to detect either presence of Antigens or Antibodies

Direct and Indirect ELISA

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Direct ELISA Indirect ELISA

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Direct ELISA

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Antibody is adsorbed onto microtiter plate

Add serum to test for antigen

Specific antigen binds if contained

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Remove test fluid and unbound antigen

Binding of antigen is strong enough to withstand rinsing

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Remove test fluid and unbound antigen

Binding of antigen is strong enough to withstand rinsing

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10

E

E

E

E

E

E

Add enzyme labeled antibody

E E

Labeled antibody binds to antigens

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EE

EE

E

E

Wash sample to remove unbound antibodies

E

E

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EE

E

Wash sample to remove unbound antibodies

E

E

E

E

E

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EE

Wash sample to remove unbound antibodies

E

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Add substrate

EEE

Measure color change

Positive Negativ

Direct and Indirect ELISA

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Direct ELISA Indirect ELISA

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Indirect ELISAAntigen is adsorbed onto microtiter plate

Add serum to test for antibody

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Antigen is adsorbed onto microtiter plate

Add serum to test for antibody

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Antigen is adsorbed onto microtiter plate

Add serum to test for antibody

Wash sample

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Antigen is adsorbed onto microtiter plate

Add serum to test for antibody

Wash sample

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E

E

E

E

E

E

E

E

Add secondary antibody

Primary antibody binds to secondary anitbody

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EEE

E

E

E

E

E

Add secondary antibody

Wash unbound antibodies

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EEE

E

E

E

E

E

Add secondary antibody

Wash unbound antibodies

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EEE

Add secondary antibody

Wash unbound antibodies

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EEE

Positive Negativ

Add substrate

Measure color change

Direct vs. Indirect ELISA

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Quick methodology – only one AB

Advantages of Indirect ELISA:

Immunoreactivity of primary antibody is not affected by labeling

Cross-reactivity of second AB eliminated

Wide variety of secondary antibodies available on the market

Signal amplification (several epitopes)

EEE

Advantages of Direct ELISA:

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Immobilized Antigen vs. Sandwich

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Mainly used for antigen detection

Mainly used for antibody detection

ImmobilizedAntigen:

SandwichELISA:

EEE

EEE

Competitive ELISA

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Step 1:Add sample

Step 2:Add antibodys

E E

E

Step 3: Washing

EEE

Step 4: Add substrate

E

Quick methodology – just one washing step

Applications

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Medical diagnostic to detect presence of antibodies in patient

Detection of potential allergens in food

HIV Test

But: high material costs

Drug tests

West Nile Virus

Test kits for eggs and peanuts

Slides by Mathias Bader and Simon Loew

Thank you

30Slides by Mathias Bader and Simon Loew