Advances in Drug Delivery

23rd of May 2011

ELISA:
Enzyme-Linked Immunosorbent Assay

Slides by Mathias Bader and Simon Loew

ELISA: Enzyme-Linked Immunosorbent Assay

ELISA detects substances with antigenic

properties (mainly proteins)

Based on enzymatic color-reaction

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Basic principle of ELISA

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

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Direct and Indirect ELISA

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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 Add enzyme labeled
E E antibody

E E Labeled antibody binds

to antigens
E E

E E

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 Wash sample to remove
unbound antibodies

E
E

E
E E

E E E

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 Wash sample to remove
E unbound antibodies

E E

E E E

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 Wash sample to remove
unbound antibodies

E E E

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 Add substrate

Measure color change

Positive Negativ

E E E

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Direct and Indirect ELISA

Direct ELISA Indirect ELISA

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 Antigen is adsorbed onto
microtiter plate
Indirect ELISA 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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 Add secondary antibody
E E

E E
Primary antibody binds
to secondary anitbody
E E

E E

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 Add secondary antibody

Wash unbound antibodies

E
E
E

E
E
E E E

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 Add secondary antibody
E
E
Wash unbound antibodies
E

E E E

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 Add secondary antibody

Wash unbound antibodies

E E E

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 Add substrate

Measure color change

Positive Negativ

E E E

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Direct vs. Indirect ELISA
Advantages of Indirect ELISA:

Immunoreactivity of primary
antibody is not affected by labeling E E E
Wide variety of secondary
antibodies available on the market
Signal amplification (several epitopes)

Advantages of Direct ELISA:

Quick methodology – only one AB

E E E
Cross-reactivity of second AB
eliminated

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Immobilized Antigen vs. Sandwich

Immobilized Mainly used for antibody

Antigen: detection E E E

Sandwich Mainly used for antigen

ELISA: detection
E E E

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Competitive ELISA

Step 1: Step 2: Step 3: Step 4:

Add sample Add antibodys Washing Add substrate

E E E

Quick methodology – just one washing step

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Applications

Medical diagnostic to detect presence of antibodies in patient

HIV Test

West Nile Virus

Detection of potential allergens in food

Test kits for eggs and peanuts

Drug tests

But: high material costs

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 Thank you

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