The document discusses enzyme-linked immunosorbent assays (ELISAs), which can qualitatively and quantitatively measure antigen-antibody binding. There are three main types of ELISAs - indirect ELISAs measure antibodies, sandwich ELISAs measure antigens, and competitive ELISAs also measure antigens but the extent of color development is inversely proportional to the amount of antigen. The document provides details on the basic procedures and components needed to perform each type of ELISA.

1. ELISAs
Enzyme Linked Immunosorbent Assays

2. Antibody-Antigen Reactions
Applications
 Agglutination reactions
 Precipitation reactions
 Western blotting/immunoblotting
 Direct and indirect immunofluorescence
 Flow Cytometry
 ELISAs

3. What the ELISA tells us

4. What the ELISA tells us
 The ELISA (Enzyme-Linked
ImmunoSorbent Assay) can be used
both qualitatively and quantitatively to
measure antigen-antibody binding.

5. What the ELISA tells us
 The ELISA (Enzyme-Linked
ImmunoSorbent Assay) can be used
both qualitatively and quantitatively to
measure antigen-antibody binding.

6. What the ELISA tells us
 The ELISA (Enzyme-Linked
ImmunoSorbent Assay) can be used
both qualitatively and quantitatively to
measure antigen-antibody binding.
 Depending on what variation is used,
ELISAs will detect antigen or antibody.

7. Applications of ELISA
Antigens detected by ELISAs include:
 hormones
 enzymes
 microbial antigens
 illicit drugs
Antibodies detected by ELISAs include:
 antibodies in body fluids
 antibodies in tissue culture supernatants
 anti-HIV in the screening test for HIV infection
 Anti-West Nile in the screening for West Nile Virus

8. Advantages of the ELISA method
The ELISA is probably the most commonly
used immunological assay because of its:
 versatility
 sensitivity (ability to detect small amounts of
antigen or antibody)
 specificity (ability to discriminate between
closely related but antigenically different
molecules)

9. What is needed to perform the assay
 Purified antigen (if you want to detect or quantify
antibody).
 Purified antibody (if you want to detect or quantify
antigen).
 Standard solutions (positive and negative controls).
 Sample to be tested.
 Microtiter dishes: plastic trays with small wells in which
the assay is done.
 Wash fluid (buffer).
 Enzyme-labeled antibody and enzyme substrate.
 ELISA reader (spectrophotometer) for quantitative
measurements.

10. What we are using
 Capture antibody: α-IL-2
 Purified antigen: IL-2
 Detection antibody: HRP conjugated α-IL-2
 Wash buffer: PBS-T
 Enzyme substrate: Stabilized 3,3’, 5,5’
Tetramethylbenzidine

11. How to interpret the results
 The amount of colored product is proportional to
the amount of enzyme-linked antibody that
binds, which is directly related to the amount of
antibody that was present to bind antigen or
antigen that was present to bind antibody.
 If known amounts of antigen or antibody are
added, a standard curve can be constructed
which will allow the amount of unknown antigen
or antibody to be determined.

12. The Indirect ELISA
Measures antibody

13. The Indirect ELISA

14. The Indirect ELISA

15. The Indirect ELISA

16. The Indirect ELISA

17. The Indirect ELISA

18. The Indirect ELISA

19. The Sandwich ELISA
Measures antigen

20. The Sandwich ELISA

21. The Sandwich ELISA

22. The Sandwich ELISA

23. The Sandwich ELISA

24. The Sandwich ELISA

25. The Sandwich ELISA

26. The Competitive ELISA
 This assay is based on the competitive binding
technique in which antigen present in a sample
competes with a fixed amount of enzyme
conjugate for binding sites on an antibody-
coated plate.
 The extent of color development is inversely
proportional to the amount of antigen in the
sample.
Measures antigen

27. The Competative ELISA

28. The Competative ELISA

29. The Competative ELISA

30. The Competative ELISA

31. The Competative ELISA

32. The Competative ELISA

33. The Competative ELISA

34. The Competative ELISA

35. Add
capture Wash
antibody
Block Wash
Dilute Aliquot Aliquot
Standard Standard Unknown
Incubate