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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
Slides by Mathias Bader and Simon Loew

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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

Slides by Mathias Bader and Simon Loew

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Direct and Indirect ELISA Direct ELISA Slides by Mathias Bader and Simon Loew Indirect ELISA 4 .

Direct ELISA Slides by Mathias Bader and Simon Loew 5 .

Antibody is adsorbed onto microtiter plate Add serum to test for antigen Specific antigen binds if contained Slides by Mathias Bader and Simon Loew 6 .

Remove test fluid and unbound antigen Binding of antigen is strong enough to withstand rinsing Slides by Mathias Bader and Simon Loew 7 .

Remove test fluid and unbound antigen Binding of antigen is strong enough to withstand rinsing Slides by Mathias Bader and Simon Loew 8 .

Slides by Mathias Bader and Simon Loew 9 .

Add enzyme labeled antibody E E Labeled antibody binds to antigens E E E E E Slides by Mathias Bader and Simon Loew E 10 .

Wash sample to remove unbound antibodies E E E E E Slides by Mathias Bader and Simon Loew E E E 11 .

Wash sample to remove unbound antibodies E E E E E E E Slides by Mathias Bader and Simon Loew E 12 .

Wash sample to remove unbound antibodies E E Slides by Mathias Bader and Simon Loew E 13 .

Add substrate Measure color change Positive E E Slides by Mathias Bader and Simon Loew Negativ E 14 .

Direct and Indirect ELISA Direct ELISA Slides by Mathias Bader and Simon Loew Indirect ELISA 15 .

Indirect ELISA Slides by Mathias Bader and Simon Loew Antigen is adsorbed onto microtiter plate Add serum to test for antibody 16 .

Antigen is adsorbed onto microtiter plate Add serum to test for antibody Slides by Mathias Bader and Simon Loew 17 .

Antigen is adsorbed onto microtiter plate Add serum to test for antibody Wash sample Slides by Mathias Bader and Simon Loew 18 .

Antigen is adsorbed onto microtiter plate Add serum to test for antibody Wash sample Slides by Mathias Bader and Simon Loew 19 .

Slides by Mathias Bader and Simon Loew 20 .

E Add secondary antibody E E Primary antibody binds to secondary anitbody E E E E Slides by Mathias Bader and Simon Loew E 21 .

Add secondary antibody Wash unbound antibodies E E E E Slides by Mathias Bader and Simon Loew E E E E 22 .

Add secondary antibody E E Wash unbound antibodies E E E E E Slides by Mathias Bader and Simon Loew E 23 .

Add secondary antibody Wash unbound antibodies E E Slides by Mathias Bader and Simon Loew E 24 .

Add substrate Measure color change Positive E E Slides by Mathias Bader and Simon Loew Negativ E 25 .

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 Cross-reactivity of second AB eliminated Slides by Mathias Bader and Simon Loew 26 E .Direct vs.

Sandwich Immobilized Antigen: Sandwich ELISA: Mainly used for antibody detection E E Mainly used for antigen detection Slides by Mathias Bader and Simon Loew E E 27 E E .Immobilized Antigen vs.

Competitive ELISA Step 1: Add sample Step 2: Add antibodys Step 3: Washing E Step 4: Add substrate E E E E E E Quick methodology – just one washing step Slides by Mathias Bader and Simon Loew 28 .

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 Slides by Mathias Bader and Simon Loew 29 .

Thank you Slides by Mathias Bader and Simon Loew 30 .