Enzyme linked immuno sorbant assay (ELISA)

1. Enzyme-Linked ImmunoSorbant
Assay (ELISA)
Presented by:
Amany ELshamy

2. Outline
• Introduction
• Principle
• Types
• Material and steps (Direct)
• Material and steps (Indirect)
• Equipment
• Application
• Advantages
• Disadvantages
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3. Introduction
• In 1971, Engvall, Pearlmann, van
Weemen and Schuurs described
the first ELISA technique.
• ELISA is an immunological assay
based on the Ag-Ab Reaction.
• ELISA is also called enzyme
immunoassay.
• It used to detect Antigens,
Antibodies, Proteins, Hormones
and Drugs.
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4. Introduction
• ELISA is a heterogeneous enzyme
immunoassay that can be either competitive
or non competitive.
• ELISA reaction can be measured in both
qualitative and quantitative terms.
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5. General Principle
• The basic principle of ELISA is antigen-
antibody reactions.
• The technique depends on the specificity and
high affinity of antibodies for their
complementary antigen.
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6. General Principle
• The basic principle of an ELISA is to use an
enzyme to detect the Ag-Ab binding.
• The enzyme converts a colorless substrate
(chromogen) to a colored product, indicating
the presence of Ag-Ab Reaction.
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7. General Application
• ELISA can diagnose infections by detecting the
presence of antigens (proteins of the
pathogen) in the patient serum or by
detecting the antibodies produced against the
pathogen.
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8. General Steps
• The four main general steps to completing an
ELISA immunoassay are:
1. Coating (with either antigen or antibody).
2. Blocking.
3. Detection.
4. Reading.
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9. General Advantages
• Sensitive and Specific.
• Inexpensive.
• Preliminary diagnostic tools.
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10. Types
The ELISA tests are of different types like:
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
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12. 1. Direct ELISA
• It is a biochemical technique used mainly in
immunology to directly detect the presence
of an antibody or an antigen in a sample.
• The direct ELISA uses the method of directly
labeling the antibody itself.
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13. 1. Coated Plate (Antigen).
2. Enzyme Conjugate-Labeled Antibody.
3. Buffers ( Coating, Washing and Blocking).
4. Substrate.
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Material of Direct ELISA

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Material of Direct ELISA

15. • Micro well plates are coated with a sample
containing the target.
• The binding of labeled antibody is quantitated by a
colorimetric, chemiluminescent, or fluorescent.
end-point.
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Steps of Direct ELISA

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Steps of Direct ELISA

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Steps of Direct ELISA
1. Apply a sample of known antigen to a
surface.
2. Enzyme linked primary antibody is applied to
the plate.
3. Washed, After this wash, only the antibody-
antigen complexes remain attached.
4. Apply a substrate which is converted by the
enzyme to elicit a chromogenic signal.

18. Equipment
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ELISA Readers: Readers need to have appropriate filter (650 nm
and 450 nm).

19. Advantages of Direct ELISA Method
• Quick methodology while minimizing potential
user error (one antibody is used).
• Less reagents and fewer steps are required
making this ELISA format simple.
• Cross-reactivity of secondary antibody is
eliminated.
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20. Disadvantages of Direct ELISA
Method
• Antigen immobilization is not specific resulting in
potentially high background interference.
• Primary antibody must be labeled individually,
which is time-consuming and expensive.
• Low flexibility – a specific conjugated primary
antibody is needed for each target protein.
• No signal amplification, which reduces assay
sensitivity.
• Immunoreactivity of the primary antibody may
be adversely affected by labeling with enzymes.
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22. 2. Indirect ELISA Principle
• It is a technique that uses a two-step process
for detection.
A. Unlabeled Primary antibody specific for the
antigen binds to the target.
B. Labeled secondary antibody against the host
species of the primary antibody binds to the
primary antibody for detection.
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23. 2. Indirect ELISA Principle
• The indirect ELISA utilizes an unlabeled
primary antibody in conjunction with a
labeled secondary antibody.
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24. 1. Coated Plate (Antigen).
2. Primary Antibody (unlabeled).
3. Enzyme-conjugated- labeled Secondary
Antibody.
4. Buffers ( Coating, Washing and Blocking).
5. Substrate.
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Material of Indirect ELISA

25. Steps
• Antigen is added to plate.
• Added Blocking buffer.
• Unlabeled primary antibody is added.
• Secondary antibody- conjugated enzyme is
then added which recognizes and binds to the
primary antibody.
• Substrate is finally added.
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26. Steps
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27. Steps
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28. Equipment
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ELISA Readers: Readers need to have appropriate filter (650 nm
and 450 nm).

29. Advantages of Indirect ELISA Method
• High sensitivity (2nd Antibody).
• Cost-effective and less expensive.
• Flexibility (different primary antibodies can be
used with a single labeled secondary
antibody).
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30. Disadvantages of indirect ELISA
• It takes Long time.
• Cross-reactivity (due to use of secondary
antibody, which could increase background
noise).
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31. When you typically use Direct or
Indirect ELISA?
Direct ELISA:
It is typically used when the Antibody to an
antigen needs to be quantified and analyzed.
The indirect ELISA:
It is suitable for determining the total antibody
concentration in samples ( It is used to detect
antibodies in patient serum by attaching antigen
to the well of a microtiter plate, allowing the
patient (primary) antibody to bind the antigen
and an enzyme-conjugated secondary antibody
to detect the primary antibody).
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32. Videos
• https://www.youtube.com/watch?v=CWkrQrq0yx
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• https://www.youtube.com/watch?v=zZasVbK37z
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• https://www.youtube.com/watch?v=amz5Xv4hl7
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• https://www.youtube.com/watch?v=kBmqgLy5E3
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• https://www.youtube.com/watch?v=GsJHYeQy5S
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