3. INTRODUCTION
ELISA (Enzyme Linked Immuno Sorbent assay) is a widely used technique for
detection of antigen(Ag) or antibody(Ab).
The technique was developed in 1971 by peter perlmon 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
in1966.
4. PRINCIPLE
Priciple is based on the formation of Ag-Ab complex, which is detected by
chromogenic detection using enzyme conjugated secondary antibody.
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. TYPES OF ELISA
Direct ELISA
Indirect ELISA
Sandwich ELISA
Competitive ELISA
6. 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 detector.
7. 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
product.
8. 3. SANDWICH ELISA
It is used for detecting an antigen in the given sample.
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.
9. 4.COMPETITIVE ELISA
This variation of ELISA is used to quantitatively estimate the amount of
antigen in the given sample.
Ag and ab are initially incubated so that they from 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.
10. APPLICATIONS
Since ELISA can detect both antigen and antibody it is a useful tool for
determining serum antibody concentrations.
It has also found applications in the food industry in detecting potential food
allergens, such as mick, peanuts, walnuts, and eggs.
The other uses of ELISA include:
Detection of Mycobacterium antibodies in tuberculosis.
Detection of hepatitis B markers in serum.
Detection of enterotoxin of E.coli in feces.
Detection of HIV antibodies in blood samples.
11. ADVANTAGES
Sensitive assay equipments are widely available.
No radiation hazards.
Reagents are cheap with long shelf life.
Qualitative and quantitative.
ELISA can used on most types of biological samples, such as plasma, serum,
urine, and cell extracts.
12. DISADVANTAGES
Only monoclonal antibodies can be used as matched pairs.
Monoclonal antibodies can cost more than polyclonal antibodies.
Negative controls ay indicate positive result 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 colour must be read as soon as
possible.
13. CONCLUSION
Enzyme linked immunosorbent assay (ELISA) is a noble 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 screening.
Chromogenic detection method used in ELISA is convenient and sensitive for
any assay and is hazard free.
14. REFERENCES
Kuby immunology by Kindt, Goldsby and Osborne
Medical immunology by Gabriel Virella
www.google.com
www.wekipedia.org
