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.

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ELISA
(Enzyme-linked Immunosorbent assay)
Introduction, Principle and Applications
INTRODUCTION:
 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.
Type:
1. Direct ELISA
2. Indirect ELISA
3. Sandwich ELISA
4. Competitive ELISA
1. Direct ELISA:
 In a direct ELISA, the antigen is immobilized to the surface of the multi-well plate and detected
with an antibody specific for the antigen.
 The antibody is directly conjugated to HRP (One commonly used enzyme conjugate
in ELISA is horseradish peroxidase. Horseradish peroxidase) or other detection molecules.
2. Indirect ELISA:
 Indirect ELISA detects the presence of an antibody in a sample.
 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.

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 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:
 Primary antibody (unlabeled) is incubated with sample antigen.
 Antibody-antigen complexes are then added to 96-well plates which are pre-coated with the
same antigen.
 Unbound antibody is removed by washing the plate. (The more antigen in the sample, the less
antibody will be able to bind to the antigen in the well, hence "competition.").
 The secondary antibody that is specific to the primary antibody and conjugated with an enzyme
is added.
 A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.

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 For competitive ELISA, the higher the sample antigen concentration, the weaker the eventual
signal.
PRINCIPLE:
 In ELISA, various antigen-antibody combinations are used, always including an enzyme-labeled antigen
or antibody, and enzyme activity is measured colorimetrically.
 The enzyme activity is measured using a substrate that changes color when modified by the enzyme.
Light absorption of the product formed after substrate addition is measured and converted to numeric
values.
Depending on the antigen-antibody combination, the assay is called a direct ELISA, indirect ELISA,
sandwich ELISA, competitive ELISA etc.
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.