Advances in Drug Delivery<br />23rd of May 2011<br />ELISA: Enzyme-Linked Immunosorbent Assay<br />Slides by Mathias Bader...
ELISA: Enzyme-Linked Immunosorbent Assay<br />2<br />ELISA detects substances with antigenic properties (mainly proteins)<...
Basic principle of ELISA<br />3<br />Enzyme is used to detect the binding of Antibody - Antigen<br />Enzyme converts color...
Direct and Indirect ELISA<br />4<br />Indirect ELISA<br />Direct ELISA<br />Slides by Mathias Bader and Simon Loew<br />
5<br />Direct ELISA<br />Slides by Mathias Bader and Simon Loew<br />
6<br />Antibody is adsorbed onto microtiter plate<br />Add serum to test for antigen<br />Specific antigen binds if contai...
7<br />Remove test fluid and unbound antigen<br />Binding of antigen is strong enough to withstand rinsing<br />Slides by ...
8<br />Remove test fluid and unbound antigen<br />Binding of antigen is strong enough to withstand rinsing<br />Slides by ...
9<br />Slides by Mathias Bader and Simon Loew<br />
10<br />Add enzyme labeled antibody<br />Labeled antibody binds to antigens<br />E<br />E<br />E<br />E<br />E<br />E<br /...
11<br />Wash sample to remove unbound antibodies<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />Slides by Ma...
12<br />Wash sample to remove unbound antibodies<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />Slides by Ma...
13<br />Wash sample to remove unbound antibodies<br />E<br />E<br />E<br />Slides by Mathias Bader and Simon Loew<br />
14<br />Add substrate<br />Measure color change<br />Positive<br />Negativ<br />E<br />E<br />E<br />Slides by Mathias Bad...
Direct and Indirect ELISA<br />15<br />Indirect ELISA<br />Direct ELISA<br />Slides by Mathias Bader and Simon Loew<br />
16<br />Antigen is adsorbed onto microtiter plate<br />Indirect ELISA<br />Add serum to test for antibody<br />Slides by M...
17<br />Antigen is adsorbed onto microtiter plate<br />Add serum to test for antibody<br />Slides by Mathias Bader and Sim...
18<br />Antigen is adsorbed onto microtiter plate<br />Add serum to test for antibody<br />Wash sample<br />Slides by Math...
19<br />Antigen is adsorbed onto microtiter plate<br />Add serum to test for antibody<br />Wash sample<br />Slides by Math...
20<br />Slides by Mathias Bader and Simon Loew<br />
21<br />Add secondary antibody<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />Primary antibody binds to seco...
22<br />Add secondary antibody<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />Wash unbound antibodies<br />S...
23<br />Add secondary antibody<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />Wash unbound antibodies<br />S...
24<br />Add secondary antibody<br />E<br />E<br />E<br />Wash unbound antibodies<br />Slides by Mathias Bader and Simon Lo...
25<br />Add substrate<br />Measure color change<br />Positive<br />Negativ<br />E<br />E<br />E<br />Slides by Mathias Bad...
Direct vs. Indirect ELISA<br />26<br />Advantages of Indirect ELISA:<br />Immunoreactivity of primary antibody is not affe...
Immobilized Antigen vs. Sandwich<br />27<br />Immobilized<br />Antigen:<br />Mainly used for antibodydetection<br />E<br /...
Competitive ELISA<br />28<br />Step 2:<br />Add antibodys<br />Step 1:<br />Add sample<br />Step 3: Washing<br />Step 4: <...
Applications<br />29<br />Medical diagnostic to detect presence of antibodies in patient <br />HIV Test<br />West Nile Vir...
Thankyou<br />30<br />Slides by Mathias Bader and Simon Loew<br />
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ELISA Test: Enzyme-linked Immunosorbent Assay

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ELISA Test: Enzyme-linked Immunosorbent Assay

  1. 1. Advances in Drug Delivery<br />23rd of May 2011<br />ELISA: Enzyme-Linked Immunosorbent Assay<br />Slides by Mathias Bader and Simon Loew<br />
  2. 2. ELISA: Enzyme-Linked Immunosorbent Assay<br />2<br />ELISA detects substances with antigenic properties (mainly proteins)<br />Based on enzymatic color-reaction<br />Slides by Mathias Bader and Simon Loew<br />
  3. 3. Basic principle of ELISA<br />3<br />Enzyme is used to detect the binding of Antibody - Antigen<br />Enzyme converts colorless substrate into colored product, indicating the presence of Antibody - Antigen complex<br />ELISA can be used to detect either presence of Antigens or Antibodies<br />Slides by Mathias Bader and Simon Loew<br />
  4. 4. Direct and Indirect ELISA<br />4<br />Indirect ELISA<br />Direct ELISA<br />Slides by Mathias Bader and Simon Loew<br />
  5. 5. 5<br />Direct ELISA<br />Slides by Mathias Bader and Simon Loew<br />
  6. 6. 6<br />Antibody is adsorbed onto microtiter plate<br />Add serum to test for antigen<br />Specific antigen binds if contained<br />Slides by Mathias Bader and Simon Loew<br />
  7. 7. 7<br />Remove test fluid and unbound antigen<br />Binding of antigen is strong enough to withstand rinsing<br />Slides by Mathias Bader and Simon Loew<br />
  8. 8. 8<br />Remove test fluid and unbound antigen<br />Binding of antigen is strong enough to withstand rinsing<br />Slides by Mathias Bader and Simon Loew<br />
  9. 9. 9<br />Slides by Mathias Bader and Simon Loew<br />
  10. 10. 10<br />Add enzyme labeled antibody<br />Labeled antibody binds to antigens<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />Slides by Mathias Bader and Simon Loew<br />
  11. 11. 11<br />Wash sample to remove unbound antibodies<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />Slides by Mathias Bader and Simon Loew<br />
  12. 12. 12<br />Wash sample to remove unbound antibodies<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />Slides by Mathias Bader and Simon Loew<br />
  13. 13. 13<br />Wash sample to remove unbound antibodies<br />E<br />E<br />E<br />Slides by Mathias Bader and Simon Loew<br />
  14. 14. 14<br />Add substrate<br />Measure color change<br />Positive<br />Negativ<br />E<br />E<br />E<br />Slides by Mathias Bader and Simon Loew<br />
  15. 15. Direct and Indirect ELISA<br />15<br />Indirect ELISA<br />Direct ELISA<br />Slides by Mathias Bader and Simon Loew<br />
  16. 16. 16<br />Antigen is adsorbed onto microtiter plate<br />Indirect ELISA<br />Add serum to test for antibody<br />Slides by Mathias Bader and Simon Loew<br />
  17. 17. 17<br />Antigen is adsorbed onto microtiter plate<br />Add serum to test for antibody<br />Slides by Mathias Bader and Simon Loew<br />
  18. 18. 18<br />Antigen is adsorbed onto microtiter plate<br />Add serum to test for antibody<br />Wash sample<br />Slides by Mathias Bader and Simon Loew<br />
  19. 19. 19<br />Antigen is adsorbed onto microtiter plate<br />Add serum to test for antibody<br />Wash sample<br />Slides by Mathias Bader and Simon Loew<br />
  20. 20. 20<br />Slides by Mathias Bader and Simon Loew<br />
  21. 21. 21<br />Add secondary antibody<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />Primary antibody binds to secondary anitbody<br />Slides by Mathias Bader and Simon Loew<br />
  22. 22. 22<br />Add secondary antibody<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />Wash unbound antibodies<br />Slides by Mathias Bader and Simon Loew<br />
  23. 23. 23<br />Add secondary antibody<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />Wash unbound antibodies<br />Slides by Mathias Bader and Simon Loew<br />
  24. 24. 24<br />Add secondary antibody<br />E<br />E<br />E<br />Wash unbound antibodies<br />Slides by Mathias Bader and Simon Loew<br />
  25. 25. 25<br />Add substrate<br />Measure color change<br />Positive<br />Negativ<br />E<br />E<br />E<br />Slides by Mathias Bader and Simon Loew<br />
  26. 26. Direct vs. Indirect ELISA<br />26<br />Advantages of Indirect ELISA:<br />Immunoreactivity of primary antibody is not affected by labeling<br />E<br />E<br />E<br />Wide variety of secondary antibodies available on the market<br />Signal amplification (several epitopes)<br />Advantages of Direct ELISA:<br />Quick methodology – only one AB<br />E<br />E<br />E<br />Cross-reactivity of second AB eliminated<br />Slides by Mathias Bader and Simon Loew<br />
  27. 27. Immobilized Antigen vs. Sandwich<br />27<br />Immobilized<br />Antigen:<br />Mainly used for antibodydetection<br />E<br />E<br />E<br />Mainly used for antigen detection<br />Sandwich<br />ELISA:<br />E<br />E<br />E<br />Slides by Mathias Bader and Simon Loew<br />
  28. 28. Competitive ELISA<br />28<br />Step 2:<br />Add antibodys<br />Step 1:<br />Add sample<br />Step 3: Washing<br />Step 4: <br />Add substrate<br />E<br />E<br />E<br />E<br />E<br />E<br />E<br />Quick methodology – just one washing step<br />Slides by Mathias Bader and Simon Loew<br />
  29. 29. Applications<br />29<br />Medical diagnostic to detect presence of antibodies in patient <br />HIV Test<br />West Nile Virus<br />Detection of potential allergens in food<br />Test kits for eggs and peanuts<br />Drug tests<br />But: high material costs<br />Slides by Mathias Bader and Simon Loew<br />
  30. 30. Thankyou<br />30<br />Slides by Mathias Bader and Simon Loew<br />

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