ELISA Test: Enzyme-linked Immunosorbent Assay

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
2
ELISA detects substances with antigenic properties (mainly proteins)
Based on enzymatic color-reaction

Basic principle of ELISA
3
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
4
Indirect ELISA
Direct ELISA

5
Direct ELISA

6
Antibody is adsorbed onto microtiter plate
Add serum to test for antigen
Specific antigen binds if contained

7
Remove test fluid and unbound antigen
Binding of antigen is strong enough to withstand rinsing

8
Remove test fluid and unbound antigen
Binding of antigen is strong enough to withstand rinsing

9

10
Add enzyme labeled antibody
Labeled antibody binds to antigens
E
E
E
E
E
E
E
E

11
Wash sample to remove unbound antibodies
E
E
E
E
E
E
E
E

12
E
E
E
E
E
E
E
E

13
E
E
E

14
Add substrate
Measure color change
Positive
Negativ
E
E
E

Direct and Indirect ELISA
15
Indirect ELISA
Direct ELISA

16
Antigen is adsorbed onto microtiter plate
Indirect ELISA
Add serum to test for antibody

17

18
Wash sample

19
Wash sample

20

21
Add secondary antibody
E
E
E
E
E
E
E
E
Primary antibody binds to secondary anitbody

22
E
E
E
E
E
E
E
E
Wash unbound antibodies

23
E
E
E
E
E
E
E
E

24
E
E
E

25
Add substrate
Measure color change
Positive
Negativ
E
E
E

Direct vs. Indirect ELISA
26
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

Immobilized Antigen vs. Sandwich
27
Immobilized
Antigen:
Mainly used for antibodydetection
E
E
E
Mainly used for antigen detection
Sandwich
ELISA:
E
E
E

Competitive ELISA
28
Step 2:
Add antibodys
Step 1:
Add sample
Step 3: Washing
Step 4:
Add substrate
E
E
E
E
E
E
E
Quick methodology – just one washing step

Applications
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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

Thankyou
30

ELISA Test: Enzyme-linked Immunosorbent Assay

by wadi_oo on Jun 02, 2011

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ELISA Test: Enzyme-linked Immunosorbent Assay Presentation Transcript