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ELISA Test Procedure (All you need to know)

Enzyme Linked Immunosorbent Assay (ELISA)

Enzyme-linked immunosorbent assay or ELISA test procedure is a common laboratory test procedure used to detect the presence of an antigen or antibody in a sample. The test is based on the principle of antibody-antigen binding.

  • ELISA is an antigen antibody reaction.
  • In 1971, ELISA was introduced by Peter Perlmann and Eva Engvall at
    Stockholm University in Sweden.
  • It is a common laboratory technique which is usually used to measure
    the concentration of antibodies or antigens in blood.
  • ELISA is a plate-based assay technique which is used for detecting and
    quantifying substances such as peptides, proteins, antibodies and
ELISA Test Procedure

Principle of ELISA Test Procedure :

  • 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
  • To detect the bound antibodies or antigens, a secondary antibody that is
    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.

Most commonly used Enzyme/Substrate


  • Orthophenylene diamine (OPD)
  • Para-nitrophenyl phosphate
  • Tetra Methylene Benzidine
    Hydrochloride (TMBH)
  • 5-amino salicyclic acid


  • Alkaline Phosphatase
  • Horse Reddish Peroxide

Blocking Protein:

  • Bovine Serum Albumin (BSA)

Types of ELISA Test Procedure :

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

1. Direct ELISA

ELISA Test Procedure

2. Indirect ELISA

3. Sandwich ELISA

Sandwich ELISA Test Procedure

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
  • The antigen-antibody mixture is then added to the microtiter 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

Procedure of Competitive ELISA :

  1. Antibody is incubated with sample containing antigen.
  2. Antigen-antibody complex are added to the microtiter 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.
Competitive ELISA Test Procedure

Comparison among ELISA Test Procedure Methods

ELISA Test Procedure comparison
ELISA Test Procedure result

Application of ELISA:

❑ Presence of antigen or the presence of antibody in a
sample can be evaluated.
❑ Determination of serum antibody concentrations in a
virus test.
❑ Used in food industry when detecting potential food
❑ Applied in disease outbreaks- tracking the spread of
disease e.g. HIV, bird flu, common, colds, cholera, STD etc.

ELISA Test Procedure
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