Elisa 1 copy.ppt

Jinu Janet Varghese
Group: 4
Tbilisi State Medical University

History
 Before the development of the ELISA, the only option
for conducting an immunoassay was radio-immunoassay,
a technique using radioactively-labeled
antigens or antibodies.
 In radio-immunoassay, the radioactivity provides the
signal, which indicates whether a specific antigen or
antibody is present in the sample.
 Radio-immunoassay was first described in a paper by
Rosalyn Sussman Yalow and Solomon Berson
published in 1960.

 When enzymes (such as peroxidase) react with
appropriate substrates (such as ABTS or 3,3’,5,5’-
tetramethylbenzidine), a change in color occurs, which
is used as a signal. However, the signal has to be
associated with the presence of antibody or antigen.
 This linking process was independently developed by
Stratis Avrameas and G. B. Pierce. Since it is
necessary to remove any unbound antibody or antigen
by washing, the antibody or antigen has to be fixed to
the surface of the container; i.e., the immunosorbent
has to be prepared. A technique to accomplish this
was published by Wide and Jerker Porath in 1966.

What is ELISA?
 ELISA stands for enzyme-linked immunosorbent
assay. It is a commonly used laboratory test to detect
antibodies in the blood.
 It is a plate-based assay designed for detecting and
quantifying substances such as peptides, proteins,
antibodies and hormones.
 This test is usually used to detect infection with HIV. It
has a high sensitivity.

96 well microtiter plate used for
ELISA

Components of
ELISA  Antibody: IgG fraction of serum purified by affinity
chromatography.
 Enzyme: Horse Radish Peroxidase (HRP) MW
44,000, glycoprotein with 4 lysine residues.
 Substrate: TMB(3,3', 5,5', tetramethylbenzidine).
The enzyme acts as a catalyst to oxidize substrate in
the presence of Hydrogen peroxide to produce a blue
colour. Reaction is stopped with dilute acid to cause
complex to turn yellow.

Principles
The sample with an unknown amount of antigen
is immobilized on a solid support either:
A) Non-specifically or,
B) Specifically
The detection antibody is added, forming a
complex with the antigen

 The detection antibody can be covalently linked to an
enzyme, or can itself be detected by a secondary antibody
which is linked to an enzyme forming a complex.
 Between each step, the plate is typically washed with a
mild detergent solution to remove any proteins or
antibodies that are not specifically bound.
 After the final wash step, the plate is developed by adding
an enzymatic substrate to produce a visible signal, which
indicates the quantity of antigen in the sample.

Types of ELISA
Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA

A specific monoclonal antibody is first adsorbed onto
the walls of a microtiter plate.
A suspension of serum or other fluid is added to test
the presence of complementary antigen.
If the antigen is present it will bind to the antibodies
that are attached to the wall.
This binding is strong enough to withstand the rinsing
of test fluid and unbound antigen.

Another aliquot of the monoclonal antibody which
carries reporter enzyme is added to indicate a color
change when the enzyme reacts with it's substrate.
Again the sample is rinsed to remove the unbound
antibodies. If antigen is present, a complex that
includes the antibody bound to well, the antigen and
the enzyme-conjugated antibody will be formed.
The enzyme's substrate is added. A color change
reveals the presence of enzyme-labeled antibody and
it's bound antigen.

 It is the one that determines whether a specific antibody( e.g.,
HIV antibody) is present in a sample such as serum. In this
case, appropriate antigen is first adsorbed to the walls of the
microtiter plate.
 Serum that may contain antibodies against the antigen is added
to the well. Antibodies present in the sample will bind to the
antigens that are adsorbed to the wall. If the sample contains
only non-specific antibodies, they will not bind to the antigen.
 Rinsing removes any antibodies that do not specifically attach
to the antigen absorbed to the wall.

 Addition of an antibody-enzyme conjugate can detect if
antibodies have attached to the antigen. These reporter
antibodies bind to immunoglobulins(generally IgG). Since the
sample contains human antibodies, we use enzyme-conjugated
anti-human IgG antibodies for the reporter.
 The present antibody binds to the adsorbed antigen and
enzyme-conjugated antibody will bind to this complex. Sample
is rinsed to removed unbound antibodies.
 Substrate for the enzyme is added and color changes indicates
the antibody reaction with the original antigen.

 Plate is coated with a capture antibody.
 Sample is added and any antigen present binds to
capture antibody.
 Detecting antibody is added and binds to antigen.
 Enzyme-linked secondary antibody is added and binds
to detecting antibody.
 Substrate is added and is converted by enzyme to
detectable form.

 Unlabeled antibody is incubated in the presence of its
antigen.
 These bound antibody/antigen complexes are then
added to an antigen-coated well.
 The plate is washed, so that unbound antibody is
removed. (The more antigen, the less antibody bind to
the antigen in the well, hence "competition.")
 The secondary antibody, specific to the primary
antibody is added. This second antibody is coupled to
the enzyme.
 A substrate is added, and remaining enzymes elicit a
chromogenic or fluorescent signal.

Applications
 Evaluates the antigen or antibody in a sample. (Eg.,
For screening donated blood for evidence of viral
contamination)
 Measuring hormone levels( HCG, LH, TSH, T3 & T4)
 Detecting potential food allergens
 In toxicology, used as a presumptive screen for certain
classes of drugs.
 Measuring “ rheumatoid factors ” and other
autoantibody in autoimmune diseases like Lupus
Erythematosus.

Advantages
 Sensitive assay Equipments are widely available.
 No radiation hazards.
 Reagents are cheap with long shelf life.
 Adaptable to automation and high speed.
 Qualitative and quantitative.
 Reproducible.
 ELISA can be used on most types of biological
samples, such as plasma, serum, urine, and cell
extracts

Disadvantages
 Only monoclonal antibodies can be used as matched
pairs
 Monoclonal antibodies can cost more than polyclonal
antibodies
 Monoclonal antibodies more difficult to find
 Negative controls may indicate positive results if
blocking solution is ineffective [secondary antibody or
antigen (unknown sample) can bind to open sites in
well]
 Enzyme/substrate reaction is short term so microwells

If the negative controls are
giving positive results:
 Contamination of the substrate solution, enzyme-labeled
antibody, control themselves.
 Inadequate rinsing of plates.
 Inadequate blocking of plates.

If no color has developed for the
positive controls or for the
samples:
 Check all reagents for dating and storage conditions.
 Microwell plates not coated properly.
 Reagents applied in wrong order or step omitted.
 Enzyme conjugate defective or
inhibited by contaminant.

If very little color has developed
for positive controls and the test
samples:
 Check the dilution of the enzyme labeled antibody.
 The concentration of the substrate.
 Wash buffer not adequately drained after every wash
step.
 Inadequate incubation times.
 Enzyme conjugate defective or inhibited by
contaminant, substrate defective or contaminated,
Micro well plates poorly coated.

 If colour has developed for the test samples but
not the positive controls :
 Check the source of positive controls, their expiry date and
storage.
 If the color can be seen, but the
absorbance is not high as expected :
 check the wave length.

Elisa 1 copy.ppt

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Elisa 1 copy.ppt