Elisa

ELISA
PRINCIPLE:
ELISA is a plate-based "Technique which is used for detecting and quantifying substances such
as proteins, antibodies and hormones."
An enzyme conjugated with an antibody reacts with colourless substrate to generate colored
products, such a substrate is called chromogenic substrate.
• A number of enzymes have been used for ELISA like alkaline phosphatase, horse radish
peroxidase and beta-galactosidase
•Specific substrate like ortho phenyldiamine (for peroxidse), paranitrophenyl phosphate for ALP
are used which are hydrolysed by enzymes to give coloured product
TYPES OF ELISA
 Indirect ELISA
 Competitive ELISA
 Sandwich ELISA
INDIRECT ELISA:
- Antibody can be detected or quantitatively determined by indirect ELISA.

-In the Technique, antigen is coated on the microtitre wells. Serum or some other sample
containing primary antibody is added to microtitre well and allowed to react with coated antigen
- serum or any free primary antibody is washed away and bound antibody to antigen is detected
by adding an enzyme conjugated secondary antibody that binds to primary antibody.
- unbound secondary antibody is then washed away and specific substrate for the enzyme is
added.
- The amount of coloured end product is measured by spectrophotometric plate readers that can
measure absorbance of all wells of 96 well plate.
Advantages:
 Increased sensitivity, since more than one labeled antibody is bound per primary antibody
 A wide variety of labelled secondary antibodies are available commercially
 Maximum immune reactivity of primary antibody is retained as it’s not labeled
 Versatile because primary antibodies can be made in one species and some labelled
secondary antibody can be used for detection
 Cost saving since fewer marked antibodies are required
 Different visualization methods can be used with some primary antibodies
Disadvantages:
 Cross reactivity may occur with secondary antibody resulting in non-specific signal.
 An extra incubation step is required in the procedure.
SANDWICH ELISA:
 Antigen can be detected by sandwich Elisa

 In this technique antibody is coated on the well
 A sample containing antigen is added to well forming antigen antibody complex.
 After the well is washed, second enzyme linked antibody specific to different epitope of
the antigens is added and allowed to react with bound antigen.
 Then after the unbound antibody is removed by washing, finally substrate is added to
plate which is hydrolyzed by enzyme to form coloured products.
Advantages:
 High specificity since 2 antibodies are used, the antigen is specifically captured and
detected
 Suitable for complex samples since antigen doesn’t require purification prior to
management.
 Flexibility and sensitivity since both direct and in direct detection methods can be used.
Disadvantages:
 Time consuming
 Expensive.
COMPETITIVE ELISA:
 Used to measure the concentration of antigen in a sample

 In this test antibody is first incubated with a sample containing antigen.
 Antigen-antibody mixture is added to the microtitre well which is coated with antigen
 The more the antigen present in the sample the less free antibody will be available to bind
to antigen coated well.
 After the well is washed enzyme conjugated secondary antibody specific for isotype of
primary antibody is added to determine the amount of primary antibody bound to well.
 The higher the concentration of antigen in the sample the lower the absorbance.
Advantages:
 High specificity since 2 antibodies are used

 High sensitivity since both direct and indirect detection methods can be used.
 Suitable for complex samples since antigen does not require purification prior to
measurement.
APPLICATIONS OF ELISA
 Presence of antigen or antibody in a sample can be evaluated

 Determination of serum antibody concentration in a virus test
 Used in food industry when detecting potential food allergies
 Applied in disease outbreaks - tracking spread of diseases EG: HIV, Bird flu, common
cold, cholera, STD etc.

Elisa

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Elisa

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ELISA

- Antibody can be detected or quantitatively determined by indirect ELISA.

 Antigen can be detected by sandwich Elisa

 Used to measure the concentration of antigen in a sample

 High specificity since 2 antibodies are used

 Presence of antigen or antibody in a sample can be evaluated

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