What Is ELISA?

ELISA is the basic assay technique, known as enzyme-linked immunosorbent assay

(also referred to as EIA: Enzyme Immunoassay) that is carried out to detect and
measure antibodies, hormones, peptides and proteins in the blood.

Antibodies are blood proteins produced in response to a specific antigen. It helps to

examine the presence of antibodies in the body, in case of certain infectious diseases.

Principle

ELISA works on the principle that specific antibodies bind the target antigen and detect
the presence and quantity of antigens binding. In order to increase the sensitivity and
precision of the assay, the plate must be coated with antibodies with high affinity. ELISA
can provide a useful measurement of antigen-antibody concentration.

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. 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.

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 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
4. Reagents needed for the testing– Concluded in the kit (coated plates,
sample diluents, controls, wash concentrate, conjugate, substrate, stop solution)
5. 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.
6. 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).
7. 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.
8. Stop solution: It stops the enzyme substrate reaction and color development.

ELISA Procedure:
ELISA is one of the easiest blood tests that can be carried out. It is rapid, quick and
requires a blood sample of the patient. The entire procedure of ELISA is mentioned
below.

 An antibody is attached to a polystyrene plate which is a solid surface and is

attracted or has an affinity towards bacteria, other antibodies and hormones.
 A microtiter coated with antigen is filled with this antigen-antibody mixture after
which free antibodies are removed by washing.
 A second antibody specific to primary antibody is added which is usually
conjugated with an enzyme.
 Free enzyme-linked secondary antibodies are removed by washing the plate.
 Finally, the substrate is added. The substrate is converted by the enzyme to form
a coloured product, which can be measured by spectrophotometry.

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Types of ELISA

1. Direct ELISA:
The direct detection method uses a primary antibody labeled with a reporter enzyme or
a tag that reacts directly with the antigen. Direct detection can be performed with an
antigen that is directly immobilized on the assay plate or with the capture assay format.
Direct detection, while not widely used in ELISA, is quite common for
immunohistochemical staining of tissues and cells.

2. Indirect ELISA
 Indirect ELISA detects the presence of an antibody in a sample.

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  The antigen is attached to the wells of the microtitre plate.

 A sample containing the antibodies is added to the antigen-coated wells for

binding with the antigen.

 The free primary antibodies are washed away and the antigen-antibody complex
is detected by adding a secondary antibody conjugated with an enzyme that can
bind with the primary antibody.

 All the free secondary antibodies are washed away. A specific substrate is added
which gives a coloured product.

 The absorbance of the coloured product is measured by spectrophotometry.

3. Sandwich ELISA
 Sandwich ELISA helps to detect the presence of antigen in a sample.

 The microtitre well is coated by the antibody.

 The sample containing the antigen is added to the well and washed to remove
free antigens.

 Then an enzyme-linked secondary antibody, which binds to another epitope on

the antigen is added. The well is washed to remove any free secondary
antibodies.

 The enzyme-specific substrate is added to the plate to form a coloured product,

which can be measured.

4. Competitive ELISA
 Competitive ELISA helps to detect antigen concentration in a sample.
 The microtitre wells are coated with the antigen.

 Antibodies are incubated in a solution having the antigen.

 The solution of the antigen-antibody complex is added to the microtitre wells. The
well is then washed to remove any unbound antibodies.
 More the concentration of antigen in the sample, lesser the free antibodies available to
interact with the antigen, which is coated in the well.

 The enzyme-linked secondary antibody is added to detect the number of primary

antibodies present in the well.
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  The concentration is then determined by spectrophotometry.

Advantages of ELISA
Following are some of the advantages of the ELISA technique:

 Results fetched from ELISA gives an accurate diagnosis of a particular disease

since two antibodies are used.
 Can be carried out for complex samples as the antigen is not required to get
purified to detect.
 It is highly responsive since direct and indirect analysis methods can be carried
out.
 It is a rapid test, yields results quickly.
 Possible detection for ELISA ranges from the quantitative, semi-quantitative,
standard curve, qualitative, calibration curve models etc.
 Easier to perform and uncomplicated process as compared to other assays
which require the presence of radioactive materials.

Applications of ELISA
The applications of ELISA are discussed below:

1. The presence of antibodies and antigens in a sample can be determined.

2. It is used in the food industry to detect any food allergens present.

3. To determine the concentration of serum antibody in a virus test.

4. During a disease outbreak, to evaluate the spread of the disease, e.g. during
recent COVID-19 outbreak, rapid testing kits are being used to determine
presence of antibodies in the blood sample.

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