ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
Introduction:
Introduction:
Eliza is very sensitive immunological technique which is usually used to
Detect or measure the concentration of antigen or antibodies in solution.The
term was Coined by Engvall and Pearlman in 1971.
ELISAs are typically performed in 96-well (or 384-well)
polystyrene plates, which passively bind antibodies and proteins. It is this
binding and immobilization of reagents that makes ELISAs so easy to design and
perform. Having the reactants of the ELISA immobilized to the microplate
surface makes it easy to separate bound from non-bound material during the assay.
This ability to wash away nonspecifically bound materials makes the ELISA a
powerful tool for measuring specific analytes within a crude preparation
A detection enzyme or other tag can be linked directly to
the primary antibody or introduced through a secondary antibody that recognizes
the primary antibody. It can also be linked to a protein such as streptavidin
if the primary antibody is biotin labeled. The most commonly used enzyme labels
are horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other enzymes
have been used as well, but they have not gained widespread acceptance because
of limited substrate options. These include β-galactosidase,
acetylcholinesterase and catalase. A large selection of substrates is available
for performing ELISA with an HRP or AP conjugate. The choice of substrate
depends upon the required assay sensitivity and the instrumentation available
for signal-detection (spectrophotometer, fluorometer or luminometer).
BASIC PRINCIPLE OF ELISA:
Most ELISA methods
developed for the detection of antigen or antibody consist of use of
corresponding antibody or antigen in question which is firmly fixed on solid
phase, such as plastic surface of polyvinyl plate or polystyrene tube. Such
systems are also called Solid Phase Immunosorbent Assay
(SPIA).
Test sample is added in
the microtitre plate, if there is presence of Ag or Ab in the test sample,
there will be Ag-Ab reactions (with immobilized Ab or Ag). Later enzyme
labelled antibody is added in the reaction mixture, which will combine with
either test antigen or Fc portion of test antibody.
The enzyme system
consists of;
1.
An enzyme: horse radish peroxidase,
alkaline phosphatase which is labelled or linked, to a specific antibody.
2.
A specific substrate:
§ o-Phenylenediamine
dihydrochloride for peroxidase
§ P
Nitrophenyl Phosphate (PNPP)- for Alkaline Phosphatase
Substrate is added
after the antigen-antibody reaction. The enzyme catalyses (usually hydrolyses)
the substrate to give a color end point (yellow compound in case of alkaline
phosphatase). The intensity of the color is proportional to the amount of
antibody or antigen present in the test sample, which can be quantified
using ELISA reader.
Salient
Features of ELISA Test
1.
ELISA test has high sensitivity and specificity
2.
The result of quantitative ELISA tests can be read visually
3.
A large number of tests can be done at one time ELISAs are designed specifically for
screening large numbers of specimens at a time, making them suitable for use in
surveillance and centralized blood transfusion services.
4.
Reagents used for ELISA are stable and can be distributed in
district and rural laboratories but as ELISAs require sophisticated equipment
and skilled technicians to perform the tests, their use is limited to certain
circumstances.
Most Commonly
used ELISA are:
Types of ELISA on
the basis of binding structure between the Antibody and Antigen.
Ø
Direct
ELISA
Ø
Indirect
ELISA
Ø
Sandwich
ELISA
Ø
Competitive
ELISA
Direct ELISA:
A direct ELISA (enzyme-linked immunosorbent assay) is a plate-based immunosorbent assay intended for the detection and quantification of a specific analyte (e.g. antigens, antibodies, proteins, hormones, peptides, etc.) from within a complex biological sample.
A direct ELISA (enzyme-linked immunosorbent assay) is a plate-based immunosorbent assay intended for the detection and quantification of a specific analyte (e.g. antigens, antibodies, proteins, hormones, peptides, etc.) from within a complex biological sample.
Indirect ELISA
Indirect ELISA is a
two-step ELISA which involves two binding process of primary antibody and
labeled secondary antibody. The primary antibody is incubated with the antigen
followed by the incubation with the secondary antibody. However, this may lead
to nonspecific signals because of cross-reaction that the secondary antibody
may bring about.
PROCEDURE
OF INDIRECT ELISA
Ø Coat the micro titer plate wells with
antigen.
Ø Block all unbound sites to prevent false
positive results.
Ø Add sample containing antibody (e.g. rabbit
monoclonal antibody) .
Ø Wash the plate, so that unbound antibody is
removed.
Ø Add secondary antibody conjugated to an
enzyme (e.g. anti- mouse IgG).
Ø Wash the plate, so that unbound enzyme-linked
antibodies are removed.
Ø Add substrate which is converted by the
enzyme to produce a colored product.
Ø Reaction of a substrate with the enzyme to
produce a colored product.
SANDWISH ELISA
The Sandwich
Enzyme-Linked ImmunoSorbent Assay (ELISA) is a sensitive and robust method
which measures the antigen concentration in an unknown sample. The antigen of
interest is quantified between two layers of antibodies: the capture and the
detection antibody. These antibodies must bind to non-overlapping epitopes on
the antigen. Either monoclonal or affinity-purified polyclonal antibodies can
be used as capture and detection antibodies, depending on cost, the dynamic
range and the sensitivity of the final assay.
PROCEDURE OF SANDWICH ELISA:
Ø
Prepared a surface to which a known
quantity of antibody is bound.
Ø
Add the antigen-containing sample to the
plate.
Ø
Wash the plate, so that unbound antigen is
removed.
Ø
Add the enzyme-linked antibodies which are
also specific to the antigen.
Ø
Wash the plate, so that unbound
enzyme-linked antibodies are removed.
Ø
Add substrate which is converted by the
enzyme to produce a colored product.
Ø
Reaction of a substrate with the enzyme to
produce a colored product.
COMPETITIVE ELISA:
Also known as inhibition ELISA or competitive
immunoassay, this assay measures the concentration of an antigen by detection
of signal interference. Each of the
previous formats can
be adapted to the competitive format. The sample antigen competes with a
reference antigen for binding to a specific amount of labeled antibody. The
reference antigen is pre-coated on a multi-well plate. The sample is
pre-incubated with labeled antibody and added to the wells. Depending on the
amount of antigen in the sample, more or less free antibodies will be available
to bind the reference antigen. This means the more antigen there is in the
sample, the less reference antigen will be detected and the weaker the signal.
Some competitive ELISA kits use labeled antigen instead of a labeled antibody. The labeled antigen and the sample antigen (unlabeled) compete for binding to the primary antibody. The lower the amount of antigen in the sample, the stronger the signal due to more labeled antigen in the well.
Some competitive ELISA kits use labeled antigen instead of a labeled antibody. The labeled antigen and the sample antigen (unlabeled) compete for binding to the primary antibody. The lower the amount of antigen in the sample, the stronger the signal due to more labeled antigen in the well.
Application of ELISA
•
Presence of antigen or the
presence of antibody in a sample can be evaluated.
•
Determination of serum
antibody concentrations in a virus test.
•
Used in food industry
when detecting potential food allergens.
•
Applied in disease
outbreaks- tracking the spread of disease e.g. HIV, bird flu, common, colds,
cholera, STD etc
References:
Ø
Anathanarayan and
Paniker’s ,Textbook of Microbiology,10th edition, Publications by
University press(India)Private limited 2017,Page No.116-118.
Ø
Prescott,Harley and
Klein’s Microbiology,Seventh edition,published by McGraw-Hill education(Asia)
2008,Page no.877-779.