ELISA

ELISA
ENZYME-LINKED IMMUNOSORBENT ASSAY

ELISA
 DEFINITION:
 The enzyme-linked immunosorbent assay (ELISA)

 is a test that uses antibodies and color change to identify a substance.
 immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a
liquid sample or wet sample.
 The binding of antigen or antibody to a solid support usually a plastic surface at the
bottom of a well in a microtiter plate or a latex plate. (immunosorbent)
HISTORY OF ELISA
 Radioimmunoassay was first described in a scientific paper by Rosalyn Sussman

Yalow and Solomon Berson published in 1960.
 In 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden, first
conceptualized and developed ELISA technique.
PRINCIPLE
 Based on Basic Immunology Response
 Lock and Key Concept:
Ø Antigen (key)
Ø Antibody (lock)
 Key fits into the lock
 Enzyme conjugate substrates bound to a secondary antibody that binds with the
antibody-antigen complex.
 It involves detection of an analyte (i.e. the specific substance whose presence is being
quantitatively or qualitatively analyzed)
APPLICATIONS
§ Because the ELISA can be performed to evaluate either the presence of antigen or antibody in a sample,
it is a useful tool for determining serum antibody concentrations (such as with the HIV test ).
§ It has also found applications in the food industry in detecting potential food allergens
 Various infections (Dengue)
 Specific disease factors (Clotting factors VII)
The other uses ELISA include,
Ø Detection of Mycobacterium antibodies in tuberculosis.
Ø Detection of rotavirus in feces.

EQUIPMENT
 Microwell plate
 Multipipette
 Washing device
 Microplate washer
TYPES OF ELISA
 On the basis of procedure;
a) Competitive ELISA
b) Non-Competitive ELISA
I. Sandwich ELISA
II. Indirect ELISA
III. Direct ELISA

COMPETITIVE ELISA
 For detecting concentration of antigen or antibody.

 Antibody coated microwell.
 Serum antigen & labeled antigen added together which will result in competition.
 Ab-Ag enzyme complex bound is inversely related to the concentration of antigen
present in sample.
 Increased serum antigen results in reduced binding of Ag-enzyme conjugate with the
antibody producing less enzyme activity & (yellow) color formation.
COMPETITIVE ELISA
COMPETITIVE ELISA
Why competitive ELISA is used?
 This ELISA technique is commonly used when only one antibody is available for the
antigen of interest. It is also suitable for detecting small antigens that cannot be bound
by two different antibodies such as in the sandwich ELISA technique.
 HIV disease virus detection is done by competitive ELISA.

DIRECT ELISA
DIRECT ELISA:
 It uses a primary labeled anti-body that react directly with the antigen.
 It can be performed with the antigen that is directly immobilized on assay plate.
 Not widely used but common for immuno-histochemical staining of cells & tissues.
DIRECT ELISA
 An antigen is immobilized in the well of an ELISA plate.
 The antigen is then detected by an antibody directly conjugated to an enzyme such as
HRP.
Why it is used?
 This ELISA technique is typically used when the immune response to an antigen
needs to be analyzed.
 Due to single antibody involve there is no cross reactivity.
DIRECT ELISA
DIRECT ELISA
Ø ADVANTAGES:
1. Direct ELISA detection is much faster than other techniques.
2. The assay is also less prone to error since fewer reagents and steps are needed.
3. Best for analyzing the immune response to an antigen.
Ø DISADVANTAGES:
1. Less activity of antibody due to enzyme linked and may not bind with antigen.
2. Less flexible.
INDIRECT ELISA
 It utilizes a primary un-labeled antibody in conjunction with a labeled secondary antibody.
 Secondary antibody has specificity for primary antibody.

 The envelop proteins are developed by recombinant technology and coated on the surface of
the of microtiter plates.
 Suspects serum is added, and unbound proteins are washed off.
Why it is used?
 The indirect ELISA is suitable for determining total antibody concentration in samples.
 This technique is used for the detection of HIV.
 Dengue and many other viral infection.
INDIRECT ELISA
Ø ADVANTAGES:
1. Economical.
2. High sensitivity.
3. Greater flexibility.
4. Best for determining total antibody concentrations in samples.
Ø DISADVANTAGES:
1. Possibility of background noise. Longer procedure than direct ELISA.
2. Additional incubation step for secondary antibody needed.
SANDWICH ELISA
 It requires the use of matched antibody pairs (capture and detection antibodies).
 Each antibody is therefore specific for a different and non-overlapping region or

epitope of the antigen.
 The capture antibody, binds the antigen that can then be detected in a direct ELISA
or in an indirect ELISA configuration.
SANDWICH ELISA
Ø ADVANTAGES:
§ High sensitivity, 2-5 minutes more sensitive than direct or indirect ELISA.
§ High specificity, two antibodies are involved in capture and detection.
§ Flexibility, both direct and indirect can be used.

Ø DISADVANTAGES:
§ Antibody optimization can be difficult.
§ Cross reactivity may occur between capture and detection antibodies.
Why this is used?
§ Sandwich ELISAs are particularly suitable for the analysis of complex samples, since the antigen does
not need to be purified prior to measurement using this method.
ADVANTAGES
 Reagents are relatively cheap and have long shelf life.
 ELISA is highly specific and sensitive.

 No radiation hazards occur during labelling or disposal of waste.
 Easy to perform and quick procedures.

 Equipment can be inexpensive and widely available.
 ELISA can be used to variety of infections.
DISADVANTAGES
 Management of enzyme activity can be more complex than measurement of activity

of some type of radioisotopes.
 Enzyme activity can be affected by plasma constituent.

 Kits are commercially available, but not cheap.
 Very specific to a particular antigen. Won’t recognize any other antigen.
 False positive/negative possible, especially with mutated/altered antigen.
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ELISA

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ELISA

ENZYME-LINKED IMMUNOSORBENT ASSAY

 The enzyme-linked immunosorbent assay (ELISA)

 Radioimmunoassay was first described in a scientific paper by Rosalyn Sussman

 Lock and Key Concept:

 Various infections (Dengue)

 Specific disease factors (Clotting factors VII)

The other uses ELISA include,

Ø Detection of Mycobacterium antibodies in tuberculosis.

Ø Detection of rotavirus in feces.

 On the basis of procedure;

II. Indirect ELISA

III. Direct ELISA

 For detecting concentration of antigen or antibody.

Why competitive ELISA is used?

 HIV disease virus detection is done by competitive ELISA.

 It utilizes a primary un-labeled antibody in conjunction with a labeled secondary antibody.

 Secondary antibody has specificity for primary antibody.

 Each antibody is therefore specific for a different and non-overlapping region or

§ Flexibility, both direct and indirect can be used.

 Reagents are relatively cheap and have long shelf life.

 ELISA is highly specific and sensitive.

 Easy to perform and quick procedures.

 Management of enzyme activity can be more complex than measurement of activity

 Enzyme activity can be affected by plasma constituent.

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