Elisa

Enzyme-Linked Immunosorbent Assay
Dr. Ghizal Fatima
Assistant Professor
Department of Biotechnology
Era University, Lucknow

Enzyme-Linked Immunosorbent Assay
The ELISA has been used as a diagnostic tool in medicine, plant
pathology, and biotechnology, as well as a quality control check
in various industries.
An enzyme conjugated with an antibody reacts with a colorless
substrate to generate a colored reaction product.
A number of enzymes have been employed for ELISA,
including alkaline phosphatase, horseradish peroxidase, and
galactosidase.

There Are Numerous Variants of ELISA
A number of variations of ELISA have been developed, allowing
qualitative detection or quantitative measurement of either
antigen or antibody.
Each type of ELISA can be used qualitatively to detect the
presence of antibody or antigen.
Alternatively, a standard curve based on known concentrations
of antibody or antigen is prepared, from which the unknown
concentration of a sample can be determined.
INDIRECT ELISA
SANDWICH ELISA
COMPETITIVE ELISA

INDIRECT ELISA
Antibody can be detected or quantitatively determined with an
indirect ELISA.
Serum or some other sample containing primary antibody
(Ab1) is added to an antigen- coated microtiter well and
allowed to react with the antigen attached to the well.
After any free Ab1 is washed away, the presence of antibody
bound to the antigen is detected by adding an enzyme-
conjugated secondary anti-isotype antibody (Ab2), which binds
to the primary antibody.
Any free Ab2 then is washed away, and a substrate for the
enzyme is added.
The amount of colored reaction product that forms is measured
by specialized spectrophotometric plate readers, which can
measure the absorbance of all of the wells of a 96-well plate in
seconds.

Indirect ELISA is the method of choice to detect the presence of
serum antibodies against human immunodeficiency virus (HIV),
the causative agent of AIDS.
In this assay, recombinant envelope and core proteins of HIV are
adsorbedas solid-phase antigens to microtiter wells.
Individuals infected with HIV will produce serum antibodies to
epitopes on these viral proteins.
Generally, serum antibodies to HIV can be detected by indirect
ELISA within 6 weeks of infection.

Direct ELISA A buffered solution of the antigen to be
tested for is added to each well of a
microtiter plate, where it is given time to
adhere to the plastic through charge
interactions.
The primary antibody with an attached
(conjugated) enzyme is added, which binds
specifically to the test antigen coating the
well.
A substrate for this enzyme is then added.
Often, this substrate changes color upon
reaction with the enzyme.
The higher the concentration of the primary
antibody present in the serum, the stronger
the color change. Often, a spectrometer is
used to give quantitative values for color
strength.

Sandwich ELISA
Antigen can be detected or measured by a sandwich ELISA.
In this technique, the antibody (rather than the antigen) is
immobilized on a microtiter well.
A sample containing antigen is added and allowed to react with
the immobilized antibody.
After the well is washed, a second enzyme- linked antibody
specific for a different epitope on the antigen is added and
allowed to react with the bound antigen.
After any free second antibody is removed by washing,
substrate is added, and the colored reaction product is
measured.

COMPETITIVE ELISA
In this technique, antibody is first incubated in solution with a
sample containing antigen.
The antigen-antibody mixture is then added to an antigen coated
microtiter well.
The more antigen present in the sample, the less free antibody
will be available to bind to the antigen-coated well.
Addition of an enzyme-conjugated secondary antibody (Ab2)
specific for the isotype of the primary antibody can be used to
determine the amount of primary antibody bound to the well as in
an indirect ELISA.
In the competitive assay, however, the higher the concentration of
antigen in the original sample, the lower the absorbance.

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
for determining serum antibody concentrations (such as with the HIV
test or West Nile virus).
It has also found applications in the food industry in detecting
potential food allergens, such as milk, peanuts, walnuts, almonds, and
eggs and as serological blood test for coeliac disease.
ELISA can also be used in toxicology as a rapid presumptive screen
for certain classes of drugs.
detection of Mycobacterium antibodies in tuberculosis
detection of rotavirus in feces
detection of hepatitis B markers in serum
detection of enterotoxin of E. coli in feces
detection of HIV antibodies in blood samples

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