ELISA
Principle And Types of Elisa
Presented by:
• Muhammad Arslan
• Babar Ali
• Abdul Wajid
• Awais
• Waqas
INTRODUCTION
• ELISA, are quantitative immunological procedure in which Ag-Ab
reaction is monitored by enzyme measurements.
• It is used to detect the antibodies in the blood.
• The ELISA test was the first screening test commonly for HIV.
• It is highly sensitive.
PRINCIPLE :
• Use an enzyme to detect the binding of Ag –Ab.
• The enzyme converts a colorless substrate to colored product.
That indicate the presence of Ag.
• It can be used to detect the presence of both Ag and Ab.
Direct ELISA
• Antigen is immobilized to the
microtiter plate wells.
• Primary labeled antibody is used.
• No secondary antibody
• Primary antibody directly binds to
the Ag.
• Enzyme-linked to the primary Ab
reacts with substrate to produce a
visible signal that can be
measured.
• In this way, the antigen is detected
Indirect ELISA
• Antigen is immobilized to the microtiter
plate wells.
• Both a primary antibody and a secondary
antibody are used.
• Secondary antibody is labeled with an
enzyme.
• Primary antibody binds to the Ag that is
immobilized to the plate.
• Enzyme-labeled secondary Ab binds to the
primary Ab which is bound to the Ag.
• Finally, the enzyme-linked to the
secondary Ab reacts with its substrate to
produce a visible signal that can be
Sandwich ELISA
• Antigen can be detected by sandwich ELISA
• Antibody is coated on the microtiter well.
• Sample containing antigen is added to the well and allowed
to react with the antibody attached to the well, forming
antigen-antibody complex.
• Washing can be done.
• Enzyme-linked antibody specific for a different epitope on
the antigen is added and allowed to react with the bound
antigen.
• Washing can be done.
• Substrate is added to the plate which is hydrolyzed by
enzyme to form colored products.
Sandwich ELISA
Competitive ELISA
• This test is used to measure the concentration of an antigen in a
sample.
• Antibody is incubated with sample containing antigen.
• Antigen-antibody complex are added to the microtitre well which
are pre-coated with the antigen.
• Wash the plate to remove unbound antibody.
• Enzyme linked secondary antibody which is specific to the primary
antibody is added.
• Wash the plate, so that unbound enzyme-linked antibodies are
removed.
• Add substrate which is converted by the enzyme into a fluorescent
signal.
Competitive ELISA
In Qualitative Elisa:
Green color usually
means a positive
reaction, but it
depends on the
specific substrate
and detection
system used.
Yellow color (often
produced when
using TMB
substrate after
stopping the
reaction with acid)
typically indicates
a strong positive
result.
No color: Negative
SEROLOGICAL
DIAGNOSTIC
TECHNIQUES
SEROLOGICAL
DIAGNOSTIC
TECHNIQUES
Lateral Flow Tests
Principle: These are
rapid, point-of-care tests
that utilize a lateral flow
device to detect the
presence of antibodies or
antigens.
Application: Commonly
used for rapid diagnosis of
infections, including
COVID-19.
Immunofluorescence Tests (IFs
or IFTs):
Principle:
These
tests use fluorescently
labeled antibodies to
detect specific antigens
or in a sample.
Application:Used for detecting
viral antibodies or antigens, and
can be adapted to various
diagnostic applications.
Agglutination Tests:
Principle:
These tests involve
the reaction of antibodies with
antigens, leading to the clumping
or agglutination of particles (like
latex or red blood cells).
Application:
Used for detecting various
antigens and antibodies,
including those associated
with infections and blood
typing.
Complement Fixation Test:
Principle: This test relies on the
binding of antibody-antigen
complexes to complement
molecules, which can then be
detected by lysis of indicator cells.
Application: Used to detect
antibodies to certain pathogens.
Western Blot:
Principle :
This technique
separates proteins by size and
then uses antibodies to detect
specific protein bands
Application:
Used for
identifying antibodies against
specific viral or bacterial
proteins.
Neutralization Tests:
Principle:
These tests assess the ability of
antibodies to neutralize the activity of a pathogen or
toxin.
Application:
Used to evaluate the effectiveness of antibodies in
preventing infection or the effects of toxins.
