ELISA- Principle, Types and Applications

Enzyme-linked immunosorbent assay (ELISA) test is the most widely used type of
immunoassay. ELISA is a rapid test used for detecting or quantifying antibody (Ab)
against viruses, bacteria and other materials or antigen (Ag). ELISA is so named
because the test technique involves the use of an enzyme system and immunosorbent.

ELISA test is being increasingly used in the detection of antigen (infectious agent) or
antibody due to its simplicity and sensitivity. It is as sensitive as radioimmunoassay
(RIA) and requires only microlitre quantities of test reagents. It has now been widely
applied in detection of a variety of antibody and antigens such as hormones, toxins,
and viruses.

Salient Features of ELISA Test

1. ELISA test has high sensitivity and specificity.
2. The result of quantitative ELISA tests can be read visually
3. A large number of tests can be done at one time.
ELISAs are designed specifically for screening large numbers of specimens at a
time, making them suitable for use in surveillance and centralized blood
transfusion services
4. Reagents used for ELISA are stable and can be distributed in district and rural
laboratories but as ELISAs require sophisticated equipment and skilled technicians
to perform the tests, their use is limited to certain circumstances.
Materials needed in ELISA Testing
1. Pipettes, washer system, ELISA plate reader: Readers, washers and pipette are
available as manual or automated system. One of the main factors affecting
equipment selection is the number and types of test samples being run.
1. ELISA Readers: Readers need to have appropriate filter (650 nm and 450 nm).
2. Pipette: Are available as fixed as well as adjustable volume as well as single
channel and multi-channel.
3. Washing system: It can be manual system that washes one row or column at a
time or semi automated systems that wash one strip or plate at a time or fully
automated systems that can process multiple plates
2. Reagents needed for the testing– Concluded in the kit (coated plates, sample
diluents, controls, wash concentrate, conjugate, substrate, stop solution)
 Coated plates: The 96-well plates are made of polystyrene and are coated with
either inactivated antigen or antibody. The function of the plate has to hold the
immobilized either antigen or antibody. Antigen or antibody present in the
sample will bind to the plate. This coating acts as the binding site for the
antibodies or antigens in the sample.
 Controls: Negative and positive controls are provided in each kit. The controls
help to normalize or standardize each plate. Controls are also used to validate
the assay and to calculate sample results. Controls might be pre-diluted and
ready to use. (Please refer to kit for specific instructions).
 Conjugates: ELISA conjugates are enzyme labeled antibodies that react
specifically to plate bound sample analytes. Unbound conjugates are washed
away after incubation and before the addition of substrate.
 Wash Concentrate: It acts as a buffered solution containing detergent to wash
unbound material from the plate. (Not all test kits have wash concentrate; in
that case distilled water can be used for washing; please refer to kit insert for
specific instructions)
 Stop solution: It stops the enzyme substrate reaction and color development.
Principle

ELISAs are typically performed in 96-well polystyrene plates. The serum is incubated
in a well, and each well contains a different serum. A positive control serum and a
negative control serum would be included among the 96 samples being tested.
Antibodies or antigens present in serum are captured by corresponding antigen or
antibody coated on to the solid surface. After some time, the plate is washed to
remove serum and unbound antibodies or antigens with a series of wash buffer. To
detect the bound antibodies or antigens, a secondary antibodies that are attached to an
enzyme such as peroxidase or alkaline phosphatase are added to each well. After an
incubation period, the unbound secondary antibodies are washed off. When a suitable
substrate is added, the enzyme reacts with it to produce a color. This color produced is
measurable as a function or quantity of antigens or antibodies present in the given
sample. The intensity of color/ optical density is measured at 450nm. The intensity of
the color gives an indication of the amount of antigen or antibody.

Types of ELISA

Frequently there are 3 types of ELISA on the basis of binding structure between the
Antibody and Antigen.

1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
 1. Direct ELISA

For direct detection, an antigen coated to a multi-well plate is detected by an antibody

that has been directly conjugated to an enzyme. This detection method is a good
option if there is no commercially available ELISA kits for your target protein.

Advantages

 Quick because only one antibody and fewer steps are used.
 Cross-reactivity of secondary antibody is eliminated.

Disadvantages

 Immunoreactivity of the primary antibody might be adversely affected by

labeling with enzymes or tags.
 Labeling primary antibodies for each specific ELISA system is time-
consuming and expensive.
 No flexibility in choice of primary antibody label from one experiment to
another.
 Minimal signal amplification.

2. Indirect ELISA

Antibody can be detected or quantitatively determined by indirect ELISA. In this

technique, antigen is coated on the microtiter well. Serum or some other sample
containing primary antibody is added to the microtiter well and allowed to react with
the coated antigen. Any free primary antibody is washed away and the bound
antibody to the antigen is detected by adding an enzyme conjugated secondary
antibody that binds to the primary antibody. Unbound secondary antibody is then
washed away and a specific substrate for the enzyme is added. Enzyme hydrolyzes the
substrate to form colored products. The amount of colored end product is measured by
spectrophotometric plate readers that can measure the absorbance of all the wells of
96-well plate.

Procedure of Indirect ELISA

1. Coat the micro titer plate wells with antigen.

2. Block all unbound sites to prevent false positive results.
3. Add sample containing antibody (e.g. rabbit monoclonal antibody) to the wells and
incubate the plate at 37°c.
4. Wash the plate, so that unbound antibody is removed.
5. Add secondary antibody conjugated to an enzyme (e.g. anti- mouse IgG).
6. Wash the plate, so that unbound enzyme-linked antibodies are removed.
7. Add substrate which is converted by the enzyme to produce a colored product.
8. Reaction of a substrate with the enzyme to produce a colored product.

Advantages

 Increased sensitivity, since more than one labeled antibody is bound per primary
antibody.
 A wide variety of labeled secondary antibodies are available commercially.
 Maximum immunoreactivity of the primary antibody is retained because it is not
labeled.
 Versatile because many primary antibodies can be made in one species and the
same labeled secondary antibody can be used for detection.
 Flexibility, since different primary detection antibodies can be used with a single
labeled secondary antibody.
 Cost savings, since fewer labeled antibodies are required.
 Different visualization markers can be used with the same primary antibody.

Disadvantages

 Cross-reactivity might occur with the secondary antibody, resulting in nonspecific

signal.
 An extra incubation step is required in the procedure.

3. Sandwich ELISA

Antigen can be detected by sandwich ELISA. In this technique, antibody is coated on

the microtiter well. A sample containing antigen is added to the well and allowed to
react with the antibody attached to the well, forming antigen-antibody complex. After
the well is washed, a second enzyme-linked antibody specific for a different epitope
on the antigen is added and allowed to react with the bound antigen. Then after
unbound secondary antibody is removed by washing. Finally substrate is added to the
plate which is hydrolyzed by enzyme to form colored products.
Procedure of sandwich ELISA

1. Prepare a surface to which a known quantity of antibody is bound.

2. Add the antigen-containing sample to the plate and incubate the plate at 37°c.
3. Wash the plate, so that unbound antigen is removed.
4. Add the enzyme-linked antibodies which are also specific to the antigen and then
incubate at 37°c.
5. Wash the plate, so that unbound enzyme-linked antibodies are removed.
6. Add substrate which is converted by the enzyme to produce a colored product.
7. Reaction of a substrate with the enzyme to produce a colored product.

Advantages

 High specificity, since two antibodies are used the antigen is specifically captured
and detected.
 Suitable for complex samples, since the antigen does not require purification prior
to measurement.
 Flexibility and sensitivity, since both direct and indirect detection methods can be
used.

4. Competitive ELISA

This test is used to measure the concentration of an antigen in a sample.

In this test, antibody is first incubated in solution with a sample containing antigen.
The antigen-antibody mixture is then 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 the antigen-coated well. After the well is washed, enzyme
conjugated secondary antibody specific for isotype of the primary antibody is added
to determine the amount of primary antibody bound to the well. The higher the
concentration of antigen in the sample, the lower the absorbance.
Procedure

1. Antibody is incubated with sample containing antigen.

2. Antigen-antibody complex are added to the microtitre well which are pre-coated
with the antigen.
3. Wash the plate to remove unbound antibody.
4. Enzyme linked secondary antibody which is specific to the primary antibody is
added.
5. Wash the plate, so that unbound enzyme-linked antibodies are removed.
6. Add substrate which is converted by the enzyme into a fluorescent signal.

Advantages

 High specificity, since two antibodies are used.

 High sensitivity, since both direct and indirect detection methods can be used.
 Suitable for complex samples, since the antigen does not require purification prior
to measurement.

Application of ELISA

1. Presence of antigen or the presence of antibody in a sample can be evaluated.

2. Determination of serum antibody concentrations in a virus test.
3. Used in food industry when detecting potential food allergens.
4. Applied in disease outbreaks- tracking the spread of disease e.g. HIV, bird flu,
common, colds, cholera, STD etc.
Term Definition

Solid phase : Usually a microtiter plate well. Specially prepared ELISA plates are
commercially available. These have an 8 ¡Á 12 well format and can be used with a
wide variety of specialized equipment designed for rapid manipulation of samples
including multichannel pipets.

Adsorption : The process of adding an antigen or antibody, diluted in buffer, so that it

attaches passively to the solid phase on incubation. This is a simple way for
immobilization of one of the reactants in the ELISA and one of the main reasons for
its success.

Washing The simple flooding and emptying of the wells with a buffered solution to
separate bound (reacted) from unbound (unreacted) reagents in the ELISA. Again, this
is a key element to the successful exploitation of the ELISA.

Antigens : A protein or carbohydrate that when injected into animals elicits the
production of antibodies. Such antibodies can react specifically with the antigen used
and therefore can be used to detect that antigen.

Antibodies : Produced in response to antigenic stimuli. These are mainly protein

in nature. In turn, antibodies are antigenic.

Antispecies antibodies : Produced when proteins (including antibodies) from one

species are injected into another species. Thus, guinea pig serum injected into a rabbit
elicits the production of rabbit anti¨Cguinea pig antibodies.

Enzyme : A substance that can react at low concentration as a catalyst to promote a

specific reaction. Several specific enzymes are commonly used in ELISA with their
specific substrates.

Enzyme conjugate : An enzyme that is attached irreversibly to a protein, usually an

antibody. Thus, an example of antispecies enzyme conjugate is rabbit antiguinea
linked to horseradish peroxidase.

Substrate : A chemical compound with which an enzyme reacts specifically. This

reaction is used, in some way, to produce a signal that is read as a color reaction
(directly as a color change of the substrate or indirectly by its effect on another
chemical).

Chromophore : A chemical that alters color as a result of an enzyme interaction with

substrate.

Stopping : The process of stopping the action of an enzyme on a substrate. It has the
effect of stopping any further change in color in the ELISA.

Reading : Measurement of color produced in the ELISA. This is quantified using

special spectrophotometers reading at specific wavelengths for the specific colors
obtained with particular enzyme/chromophore systems. Tests can be assessed by eye.