Enzyme linked immunosorbent assay

1. ELISA
Enzyme-Linked Immunosorbent Assay
Lect: Mr.Abdul Haq
BS MLT (KMU)
M.Phil Microbiology (AUP)

2. ELISA
• An enzyme-linked immunosorbent assay, also
called ELISA or EIA, is a test that detects and
measures antibodies in your blood.
This test can be used to determine if you have
antibodies related to certain infectious
conditions
2

3. ELISA: Enzyme-Linked Immunosorbent Assay
3
ELISA detects substances with antigenic
properties (mainly proteins)
Based on enzymatic color-reaction

4. Basic principle of ELISA
4
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

5. Direct and Indirect ELISA
5
Direct ELISA Indirect ELISA

6. 6
Direct ELISA

7. 7
Antibody is adsorbed
onto microtiter plate
Add serum to test for
antigen
Specific antigen binds if
contained

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

9. 9
Remove test fluid and
unbound antigen
Binding of antigen is
strong enough to
withstand rinsing

10. 10

11. 11
E
E
E
E
E
E
Add enzyme labeled
antibody
E E
Labeled antibody binds
to antigens

12. 12
E
E
E
E
E
E
Wash sample to remove
unbound antibodies
E
E

13. 13
E
E
E
Wash sample to remove
unbound antibodies
E
E
E
E
E

14. 14
E
E
Wash sample to remove
unbound antibodies
E

15. 15
Add substrate
E
E
E
Measure color change
Positive Negativ

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

17. 17
Antigen is adsorbed onto
microtiter plate
Add serum to test for
antibody

18. 18
Antigen is adsorbed onto
microtiter plate
Add serum to test for
antibody
Wash sample

19. 19
Antigen is adsorbed onto
microtiter plate
Add serum to test for
antibody
Wash sample

20. 20

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

22. 22
E
E
E
E
E
E
E
E
Add secondary antibody
Wash unbound antibodies

23. 23
E
E
E
E
E
E
E
E
Add secondary antibody
Wash unbound antibodies

24. 24
E
E
E
Add secondary antibody
Wash unbound antibodies

25. 25
E
E
E
Positive Negativ
Add substrate
Measure color change

26. Direct vs. Indirect ELISA
26
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)
E
E
E
Advantages of Direct ELISA:
E
E
E

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

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

29. Applications
29
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

30. Thank you
30