elisa test: enzyme-linked immunosorbent assay
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ELISA: Enzyme-Linked Immunosorbent Assay
Advances in Drug Delivery23rd of May 2011
Slides by Mathias Bader and Simon Loew
ELISA: Enzyme-Linked Immunosorbent Assay
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ELISA detects substances with antigenic properties (mainly proteins)
Based on enzymatic color-reaction
Slides by Mathias Bader and Simon Loew
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
Slides by Mathias Bader and Simon Loew
Direct and Indirect ELISA
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Direct ELISA Indirect ELISA
Slides by Mathias Bader and Simon Loew
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Direct ELISA
Slides by Mathias Bader and Simon Loew
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Antibody is adsorbed onto microtiter plate
Add serum to test for antigen
Specific antigen binds if contained
Slides by Mathias Bader and Simon Loew
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Remove test fluid and unbound antigen
Binding of antigen is strong enough to withstand rinsing
Slides by Mathias Bader and Simon Loew
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Remove test fluid and unbound antigen
Binding of antigen is strong enough to withstand rinsing
Slides by Mathias Bader and Simon Loew
9Slides by Mathias Bader and Simon Loew
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E
E
E
E
E
E
Add enzyme labeled antibody
E E
Labeled antibody binds to antigens
Slides by Mathias Bader and Simon Loew
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EE
EE
E
E
Wash sample to remove unbound antibodies
E
E
Slides by Mathias Bader and Simon Loew
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EE
E
Wash sample to remove unbound antibodies
E
E
E
E
E
Slides by Mathias Bader and Simon Loew
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EE
Wash sample to remove unbound antibodies
E
Slides by Mathias Bader and Simon Loew
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Add substrate
EEE
Measure color change
Positive Negativ
Slides by Mathias Bader and Simon Loew
Direct and Indirect ELISA
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Direct ELISA Indirect ELISA
Slides by Mathias Bader and Simon Loew
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Indirect ELISA
Antigen is adsorbed onto microtiter plate
Add serum to test for antibody
Slides by Mathias Bader and Simon Loew
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Antigen is adsorbed onto microtiter plate
Add serum to test for antibody
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
Antigen is adsorbed onto microtiter plate
Add serum to test for antibody
Wash sample
Slides by Mathias Bader and Simon Loew
20Slides by Mathias Bader and Simon Loew
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E
E
E
E
E
E
E
E
Add secondary antibody
Primary antibody binds to secondary anitbody
Slides by Mathias Bader and Simon Loew
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EEE
E
E
E
E
E
Add secondary antibody
Wash unbound antibodies
Slides by Mathias Bader and Simon Loew
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EEE
E
E
E
E
E
Add secondary antibody
Wash unbound antibodies
Slides by Mathias Bader and Simon Loew
24
EEE
Add secondary antibody
Wash unbound antibodies
Slides by Mathias Bader and Simon Loew
25
EEE
Positive Negativ
Add substrate
Measure color change
Slides by Mathias Bader and Simon Loew
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:
EEE
Slides by Mathias Bader and Simon Loew
Immobilized Antigen vs. Sandwich
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Mainly used for antigen detection
Mainly used for antibody detection
ImmobilizedAntigen:
SandwichELISA:
EEE
EEE
Slides by Mathias Bader and Simon Loew
Competitive ELISA
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Step 1:Add sample
Step 2:Add antibodys
E E
E
Step 3: Washing
EE
E
Step 4: Add substrate
E
Quick methodology – just one washing step
Slides by Mathias Bader and Simon Loew
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
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