The Enzyme-Linked Immunosorbent Assay (ELISA) is a widely used biochemical technique designed to detect and quantify the presence of specific proteins, antigens, antibodies, or hormones in a sample. It relies on the principle of antigen–antibody interaction, where antibodies specifically recognize and bind to their corresponding antigens. This binding is then visualized and measured using an enzyme-linked secondary antibody that produces a detectable color change when exposed to a suitable substrate.

1. ELISA
Enzyme-Linked ImmunoSorbent Assay
By Makanju Supreme

2. Page Outline
Page 1. Title
Page 2. Page Outline
Page 3. Overview
Page 4-8. Elisa as an Immunoassay
Page 9-13. Types of Elisa
Page 14. Equipments used for Elisa and their functions
Page 15-17. How the sandwich elisa technique works
Page 18. What type of result Elisa gives
Page 19. Conclusion
Page 20. Appreciation

3. OVERVIEW
ELISA, the Enzyme-linked ImmunoSorbent Assay, is a commonly used
analytical biochemistry assay that was first described by Eva Engvall and Peter
Perlmann in 1971. It is a solid-phase type of enzyme immunoassay that detects
the presence of a ligand in a liquid sample, like plasma, using antibodies
directed against the ligand to be measured.
As earlier said and as the name clearly denotes, ELISA is an Immunoassay. It is
in fact considered the gold standard of immunoassays. It is used to diagnose a
wide range of conditions like HIV, lyme disease and even pregnancy.

4. ELISA AS AN
IMMUNOASSAY

5. ELISA AS AN IMMUNOASSAY
WHAT ARE IMMUNOASSAYS
Immunoassays are tests that rely on the interaction between antigens and
antibodies in a laboratory setting [not within the human body]
Antibodies are substances the immune system makes, that bind to unwanted
substances in order to eliminate them from the human body while Antigens are
proteins or sugars on the surface of several types of cells like viruses, proteins,
tumors and bacteria. The antigens are like markers that the antibodies can
recognise.

6. ELISA AS AN IMMUNOASSAY CONTD
For immunoassays, laboratory scientists use antigens
or antibodies they have in the lab to check for the
presence of certain antigens or antibodies in the
bodily fluid sample (like a plasma from a blood
sample).
For example, if the plasma contains antibodies for
HIV, they’ll bind to the antigen (the virus, in this case).
If the plasma sample to be tested doesn’t contain
antibodies for HIV, nothing happens.

7. ELISA AS AN IMMUNOASSAY CONTD
The laboratory scientist then adds another antibody that “knows” the HIV
antibodies. They bind to any antibody that’s already attached to the antigen.
This second antibody is linked with an enzyme (a substance that speeds up a
chemical reaction). This is the “enzyme-linked” portion of ELISA.

8. ELISA AS AN IMMUNOASSAY CONCLUSION
In the final step, the laboratory scientist adds a
substance that reacts with the enzyme. This
makes the substances change color if the
antibodies are present.
The intensity of the color change is proportional
to the amount of the antibody. So ELISA can
determine both the presence of the antibody
and how much of it there

9. TYPES OF ELISA
1.Direct
2.Indirect
3.Sandwich
4.Competitive

10. DIRECT ELISA
•Principle: An antigen is immobilized on the plate, and an enzyme-conjugated
primary antibody directly binds to it for detection.
•Best for: Detecting antigens or when a single antibody needs to be labeled.
•Advantages: Simple and quick, with no cross-reactivity from secondary
antibodies.
•Disadvantages: Lower sensitivity and flexibility because the primary antibody
must be labeled.

11. INDIRECT ELISA
•Principle: After an antigen is immobilized on the
plate, a primary antibody binds to it. An enzyme-linked secondary antibody then
binds to the primary antibody, allowing for detection.
•Best for: Detecting antibodies in a sample.
•Advantages: High sensitivity, more economical and it is flexible as the
secondary antibody can be used with different primary antibodies.
•Disadvantages: A longer protocol and potential cross-reactivity from the
secondary antibody.

12. SANDWICH ELISA
•Principle: This is the most common and most widely used form of Elisa. The
plate is first coated with a capture antibody that binds the antigen. The antigen
then binds to the capture antibody, and a second, enzyme-linked detection
antibody binds to a different epitope on the antigen, forming a "sandwich".
•Best for: Detecting antigens in complex samples.
•Advantages: High specificity and sensitivity because two antibodies target
different parts of the antigen.
•Disadvantages: Requires careful selection of two different antibodies that can
work well together.

13. COMPETITIVE ELISA
•Principle: The sample antigen competes with a labeled antigen (or antibody)
for binding to a limited number of antibody sites on a pre-coated plate.
Best for: Detecting small antigens or measuring the concentration of
antigens/antibodies by comparing binding
•Advantages: Highly sensitive for small antigens.
Disadvantages: Less commonly used than the other types

14. EQUIPMENTS USED FOR ELISA
AND THEIR FUNCTIONS
1. The Sample - This contains our target antigens to be identified
2. The Reagents - These include the enzymes[secondary antibody]
3. The Microplate - This is where the microwells fit into. i.e, where they sit.
4. The Microwell - This is where the reaction between the antigen and
antibody takes place. i.e, where the antigen-antibody complex is formed.
5. The Micropipette - This is used to pick the samples and reagents from their
bottles and drop it into the microwell.
6. The Washer - This is used to wash off unbonded excesses.
7. The microplate Reader - This is used to read the intensity of the colour
change observed.

15. HOW THE ELISA
TECHNIQUE WORKS

16. HOW THE SANDWICH ELISA
TECHNIQUE WORKS
1. Capture Antibody Coating - The microplate is coated with a capture
antibody specific to the antigen of interest
2. Washing - Unbound antibodies are washed away to prevent non-specific
binding.
3. Antigen Addition - The sample containing the antigen is added, and the
antigen binds to the immobilised captured antibody.
4. Washing - Any unbound sample components are washed away.
5. Enzyme-Linked Detection Antibody - An antibody linked to an enzyme is
added, This antibody binds to the antigen, forming a ‘sandwich.’

17. HOW THE SANDWICH ELISA
TECHNIQUE WORKS
6. Washing - Unbound enzyme-linked antibody is washed away.
7. Substrate Addition - A substrate that reacts with the enzyme is added.
8. Detection - The enzyme catalyses a reaction with the substrate to produce a
detectable signal, usually a color change.
9. Measurements - The intensity of the colour is measured by a plate reader,
which is proportional to the amount of antigen present in the sample.

18. WHAT TYPE OF RESULT DOES ELISA GIVE
Many ELISA tests have a positive or negative result, but some might be invalid:
•Positive result: This means that the test detected the substance it was
checking for.
•Negative result: This means that the test didn’t detect any of the substance it
was checking for.
•Invalid result: This means there was an error in the testing. This could mean
an issue with the sample collection or the test itself. An

19. In conclusion, even though Elisa is the gold standard of immunoassays and it
is widely used to detect diseases and illness through the absence or
presence of proteins, hormones and glycoproteins in the patient’s sample, it
is merely used as a screening procedure.
Additional tests are required and carried out by health providers to confirm
diagnosis and decide the next course of action.

20. THANK YOU FOR
YOUR TIME.