ENZYME LINKED

IMMUNOSURBENT ASSAY
ELISA

Mohammed Mufarreh
Alaa Moawia Ahmed
 Outlines
 What is ELISA
 Application
 Methods
 Types of ELISA
 Advantages & Disadvantages
What is ELISA?
 Enzyme-Linked ImmunoSorbent Assay, or ELISA, is a
biochemical technique used mainly in immunology to
detect the presence of an antibody or an antigen in a
sample.
 Enzyme-linked immunosorbent assay

Name suggests three components

– Antibody
• Allows for specific detection of analyte of interest

– Solid phase (sorbent)

• Allows one to wash away all the material that is not
specifically captured

– Enzymatic amplification
• Allows you to turn a little capture into a visible color
change that can be quantified using an absorbance
plate reader
Applications
 Because the ELISA can be performed to evaluate either
the presence of antigen or the presence of antibody in a
sample, it is a useful tool both for determining serum
antibody concentrations (such as with the HIV test or
West Nile Virus) and also for detecting the presence of
antigen.

 It has also found applications in the food industry in

detecting potential food allergens such as milk, peanuts,
walnuts, almonds, and eggs.
 What is it used for?

Measure antibody levels (allergies, vaccines).

Detect viruses (hepatitis, HIV, venereal diseases).

Detect hormonal changes (pregnancy).

Detect circulatory inflammatory markers

(cytokines)
 Materials needed
•Testing sample
•Antibody (1st, 2nd) / Antigen
•Polystyrene microtiter plate
•Blocking buffer
•Washing buffer
•Substrate
•Enzyme
Methods
1. Coat solid phase with
antigen when analysing antibody
antibody when analysing antigen

Analyte = antibody Analyte = antigen

Incubate, wash
2. Block free binding sites. Incubate. Wash.

Analyte = antibody Analyte = antigen

3. Add sample. Incubate. Wash

Analyte = antibody Analyte = antigen

4. Add conjugate. Incubate. Wash.

E E E E

Analyte = antibody Analyte = antigen

5. Add substrate
6. Incubate, stop, measure colour change

ENZYME

Colourless

OD

CONCENTRATION
Types of ELISA
Direct ELISA
Indirect ELISA
Sandwich ELISA
The ELISA plate is coated with Antibody to detect
specific antigen
Competitive ELISA
December 3, 2022 Total slides : 115 28
Microplate Washers
Micro-plate Reader

Total slides : 115

December 3, 2022 Total slides : 115 33
 Advantages of ELISA
Sensitive: nanogram levels or lower
Reproducible
Minimal reagents
Qualitative & Quantitative
Qualitative  E.g. HIV testing
quantitative assays  E.g. Drug Monitoring
Wells can be coated with Antigens OR Antibodies
Suitable for automation high speed
NO radiation hazards
 Limitations
• Results may not be absolute
• False positive possible
• False negative possible
• Antibody must be available