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ELISA (Enzyme Linked ImmunoSorbent Assay)

Presented by: Poornima R.

CONTENTS Introduction Principle Types Applications Advantages and disadvantages Summary Conclusion References

INTRODUCTION ELISA(Enzyme Linked ImmunoSorbent assay)

is a widely used technique for detection of antigen (Ag) or antibody(Ab).

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 Principle 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.

ELISA microplate reader

ELISA kits are commercially available, which can be conveniently used for laboratory

purpose.

Kit from REAGEN

Kit from Forsight

TYPES OF ELISA

1. Direct ELISA

2. Indirect ELISA

3. Sandwich ELISA

4. Competitive ELISA

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.

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.

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.

SANDWICH ELISA

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 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.

COMPETITIVE ELISA

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 milk, peanuts, walnuts, almonds, and eggs.

The other uses of ELISA include:

a. detection of Mycobacterium antibodies in tuberculosis

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

CONT...

CONT... ELISA in immunochromatography to

detect HGC in urine sample:

ADVANTAGES Sensitive assay Equipments are

widely available.

No radiation hazards.

Reagents are cheap with long

shelf life.

Qualitative and quantitative.

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

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 possible.

SUMMARYCoat plate with Ag/Ab was

h

Add blocking buffer

wash

Add test samplewash

Add enzyme conjugated Antibody

wash

Add substrate Add stop solution

Read absorbance at 450nm.

CONCLUSION 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 screening.

Chromogenic detection method used in ELISA is convenient and sensitive for any assay and is hazard free.

REFERENCES

Kuby Immunology by Kindt, Goldsby and Osborne

Medical Immunology by Gabriel Virella

www.google.com www.wekipedia.org