ELISA
GROUP 8
5/18/2022
1
OUTLINE
• Introduction
• Basic principles of ELISA
• Antigen and Antibody
• Instrumentation
• Steps in performing ELISA test
• Types of ELISA
• Applications of ELISA
5/18/2022
2
INTRODUCTION
• ELISA is an acronym of Enzyme Linked
Immunosorbent Assay
• ELISA is also called Enzyme Immuno Assay (EIA) or
Solid Phase Immuno Asssay (SPIA)
• It is a biochemical technique used mainly in
immunology to detect the presence of an
antibody or antigen in a sample
5/18/2022
3
Basic principle of ELISA
• ELISA is based on the Lock and Key concept
• An enzyme is used to detect the binding of an
antigen (Ag) – antibody (Ab)
• The enzyme converts a colorless substrate to a
colored product, indicating the presence of Ag:Ab
binding
• It can be use to detect the presence of antigen or
antibody in a sample depending on how the test is
designed
5/18/2022
4
Antigen (Ag)
 It is any molecule that induces the production of
antibodies when introduced into the body
 It is anything foreign to the immune system
 Examples include;
• Bacteria
• Viruses
• Allergens, etc.
Symbol for antigen
5/18/2022
5
Antibody (Ab)
• Antibodies are proteins produced by the immune
system which help defend against antigens
Symbol for antibody
5/18/2022
6
Instrumentation
• Microwell Plate: it is a flat bottom polystyrene
plate, containing 8 × 12 “wells” used as small test
tubes.
5/18/2022
7
• Multichannel pipette: It is used for filling
numerous vials at ones
• An 8-channel 100μL pipette is a good help for even
small-scale work
5/18/2022
8
• Microplate washer: This instrument is used for the
washing of samples from sample wells on
microplates and to prepare them for the next step
in an assay or procedure.
5/18/2022
9
ELISA reader: Also known as microplate reader or
microplate photometer
• It measures and quantitates the colour difference
in the wells of the plate.
5/18/2022
10
TYPES OF ELISA
• Direct ELISA
• Indirect ELISA
• Sandwich ELISA
• Competitive ELISA
5/18/2022
11
Direct ELISA
5/18/2022
12
Direct ELISA
• In direct ELISA, secondary antibody is not needed as
the primary antibody is used as the detection
antibody
• After coating the surface of the microwell plate with
the antigen, the primary antibody is added with the
enzyme conjugate.
• The unbound primary antibodies are washed and
then the substrate is added which is converted to
product by the enzyme to elicit
chromogenic/fluorescent signal
• The result is then quantified using ELISA reader or
spectrophotometer or any other optical device
5/18/2022
13
Indirect ELISA
5/18/2022
14
Indirect ELISA
 The sample containing the primary antibody is added to an
antigen coated microtiter well with blocking buffer and
allowed to react with the antigen attached to the well.
 Free primary antibodies are washed and the antibody
bound to antigens are detected by adding an enzyme
conjugated secondary antibody that binds to the primary
antibody.
 Free secondary antibodies are washed and the substrate
for the enzyme is added.
 The enzyme converts the substrate to product eliciting a
chromogenic/fluorescent signal which is quantified by
using ELISA reader or spectrophotometer.
5/18/2022
15
Sandwich ELISA
5/18/2022
16
Sandwich ELISA
• The surface of the microtiter plate is first coated
with a capture antibody and blocking buffer is
added to block other binding sites.
• The sample containing the antigen is added for
the antigen to bind the antibodies
• A detecting antibody is added and it binds to the
antigen.
• A secondary antibody conjugated with enzyme is
added to bind the detecting antibody
• A substrate is then added and it is converted to a
chromogenic/fluorescence product which can be
detected by ELISA reader..
5/18/2022
17
Competitive ELISA
5/18/2022
18
Competitive ELISA
• This is a very sensitive method for the
measurement of the amount of antigens
• The microtiter plate is coated with antibody
• A serum antigen and a labelled antigen are
together added
• Competition occurs between the two antigens for
the same antibody on the surface
• A substrate is added and the appearance of color
indicates a negative test whiles the absence of
color indicates a positive test.
5/18/2022
19
Applications of ELISA
• The presence of antigen or the presence of
antibody in a sample can be evaluated
• Determination of serum antibody concentration in
virus test
• Used in food industry when detecting potential
food allergens
• Applied in disease outbreaks – tracking the spread
of diseases e.g HIV, bird flu, common cold, STDS,
coronavirus,etc.
5/18/2022
20
REFERENCES
• https://www.sepmag.eu/blob/elisa-reader\
• Kuby, J., Osborne, B., A. and Goldsby, R., A.
Immunology, 5th edition, pp 148-150
• Lequin, R., M. (2005). Enzyme Immunoassay
(EIA)/Enzyme-Linked Immunosorbent Assay (ELISA).
Clinical chemistry.
5/18/2022
21
Thank you
5/18/2022
22