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By Levi Clancy for Student Reader on

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Enzyme-Linked Immunosorbent Assay (ELISA) detects presence of an antibody or antigen in an aliquot.

Two antibodies are used: one is specific to antigen, the other is coupled to an enzyme. To determine the concentration of an antibody or presence of an antigen:

  • Apply sample of known antigen to microtiter plate.

  • Antigen is fixed to surface.

  • Coat surface with serum sampled of unknown antibody concentration.

  • Plate is washed to remove unbound antibody.

  • Only antibody-antigen complexes remain attached.

  • 2nd antibodies added to wells, and bind to any antigen-antibody complexes. These are coupled to the substrate-modifying enzyme.

  • Wash plate to remove excess unbound antibodies.

  • Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorescent signal.

  • View/quantify the result using a spectrophotometer or other optical device.

  • The enzyme acts as an amplifier: even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules.

Direct Method Steps

  • Coat a plate with the protein in question

  • Wash, block, and wash the plate

  • Add an antibody to the protein

  • Wash, block, and wash the plate

  • Put on an antibody for the first antibody that fluoresces or is radioactive

  • Wash the plate

  • Add the fluorescing substrate

  • Read the plate

ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation is often used to distinguish positive and negative samples. In quantitative ELISA, the optical density or fluorescent units of the sample is interpolated into a standard curve which is typically a serial dilution of the target.

Sandwich ELISA

A less-common variant of this technique, called "sandwich" ELISA, is used to detect sample antigen. The steps are as follows:

  • Coat a plate with a known quantity of antibody for the protein you want to find

  • Wash, block and wash the plate. This removes excess antibody.

  • Apply the antigen-containing sample to the plate (apply the protein in question).

  • Wash, block and wash the plate. This removes unbound antigen.

  • Apply the enzyme-linked antibodies which are also specific to the antigen. In other words, put on another antibody for the protein that binds at a different epitope that fluoresces or is radioactive.

  • Wash the plate, so that unbound enzyme-linked antibodies are removed.

  • Add the fluorescing substrate, a chemical which is converted by the enzyme into a fluorescent signal.

  • View the result: if it fluoresces, then the sample contained antigen.

Competitive ELISA

A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different than the first two examples:

  • Unlabeled antibody is incubated in the presence of its antigen.

  • These bound antibody/antigen complexes are then added to an antigen coated well.

  • The plate is washed, so that unbound antibody is removed. (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, specific to the primary antibody is added. This second antibody is coupled to the enzyme.

  • A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.

For competitive ELISA, the higher the original antigen concentration, the weaker the eventual signal.


Based on ELISA, ELISPOT the difference between the two is that in ELISA, the substance containing the "unknown" is stuck at the bottom of the well, whereas in ELISPOT the substance with the "unknown" is placed in the well after the bottom of the well has been coated with cytokine-specific antibody. In both cases, the wells are typically contained within a generic microtiter plate. The ELISPOT method is most often used to determine the amount (i.e. the concentration) of activated antigen-specific cytotoxic T-cells in a given sample of splenocytes harvested from immunized animals, usually mice.

ELISA analysis

  • Make a standard concentration curve and fit a linear regression. Then interpolate to find the concentration of the protein. These values can be used in ANOVA or in disease modeling – just like any of the other data (either in a binomial or linear format).