ELISA(Enzyme Linked ImmunoSorbent assay) is a widely
used technique for detection of antigen (Ag) or
The technique was developed in 1971 by Peter Perlmann
and Eva Engvall at Stockholm University, Sweden.
A technique to prepare something like immunosorbent to
fix antibody or antigen to the surface of a container was
published by Wide and Jerker Porath in 1966
Eva Engvall Peter Perlmann
Principle is based on the formation of Ag-Ab
complex , which is detected by chromogenic
detection using enzyme conjugated secondary
The conjugated enzyme acts on a specific substrate
called chromogenic substrate, and generates a
coloured reaction product.
This product is qualitatively or quantitatively read
using an ELISA plate reader.
5. ELISA kits are commercially available, which can
be conveniently used for laboratory purpose.
Kit from REAGEN
Kit from Forsight
6. TYPES OF ELISA
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
7. 1.DIRECT ELISA
It is used in the detection of antigen
in the given biological sample.
Microtiter wells are initially coated
with antigen to be detected which is
followed by an antibody linked to an
enzyme conjugate. This follows the
addition of substrate which produces
colour detected using ELISA
9. 2. INDIRECT ELISA
It is used for detection of an
antibody in the given sample.
Microtiter wells are initially coated
with antigen specific for antibody to
be detected, followed by the
addition of sample. Enzyme
conjugated Secondary Antibody is
added followed by the substrate
which forms a coloured reaction
11. 3.SANDWICH ELISA
It is used for detecting an antigen in the
Microtiter wells are initially coated with
monoclonal antibodies(called capture
antibody) raised against antigen to be
detected, followed by addition of sample.
Any trace of antigen is detected by adding
primary antibody (a MAb),followed by
enzyme conjugated secondary Ab and a
chromogenic substrate; or by directly
adding an enzyme conjugated primary Ab.
13. 4.COMPETITIVE ELISA
This variation of ELISA is used to quantitatively
estimate the amount of antigen in the given
Ag and Ab are initially incubated so that they
form Ag-Ab complex. This mixture is then
added to microtiter wells coated with
synthetic analogue of antigen to be detected,
any free antibody binds to these antigens .
This complex is estimated by enzyme
conjugated secondary antibody by
chromogenic detection .More the amount of
antigen in the sample, lesser is the antibody
available to bind to microtiter wells.
Since ELISA can detect both antigen and antibody
it is a useful tool for determining serum antibody
It has also found applications in the food industry
in detecting potential food allergens, such
as milk, peanuts, walnuts, almonds, and eggs.
The other uses of ELISA include:
a. detection of Mycobacterium antibodies in
b. detection of hepatitis B markers in serum
c. detection of enterotoxin of E. coli in feces
d. detection of HIV antibodies in blood samples
Sensitive assay Equipments are
No radiation hazards.
Reagents are cheap with long shelf
Qualitative and quantitative.
ELISA can be used on most types of
biological samples, such as plasma,
serum, urine, and cell extracts
Only monoclonal antibodies can be used
as matched pairs
Monoclonal antibodies can cost more than
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
hence color must be read as soon as
with Ag/Ab wash
Add blocking buffer
Add test sample
Add substrate Add stop solution
Enzyme Linked ImmunoSorbent Assay
(ELISA) is a novel technique useful in
detecting (qualitatively and
quantitatively) an antigen or antibody
present in the given biological sample.
Besides its disadvantages the technique is
being widely used in diagnostics and drug
Chromogenic detection method used in
ELISA is convenient and sensitive for any
assay and is hazard free.