ELISA Assay Technology

Applications & Techniques

ELISA Assay Technology is a versatile immunoassay technique, a  immunoenzymatic tests, is ideal for detecting and quantifying a specific molecule in a complex sample.

The enzyme-linked immunosorbent assay (ELISA), also called enzyme immunoassay (EIA), is an effective method for detecting and quantifying the presence of specific substances, commonly proteins (or peptides, antibodies, and hormones) in a complex liquid mixture, using antibodies. 

The ELISA Technology is based on the use of antibodies labeled with an enzyme (generally peroxidase or alkaline phosphatase), so that the resulting conjugates have both immunological and enzymatic activity. The antigen-antibody reaction results immobilized because one of the components (antigen or antibody) has been previously adhered to the plate. This reaction is easily highlighted with the addition of the substrate which, reacting with the enzyme, will produce a color that is visible and quantifiable with the spectrophotometer.

ELISA Assay is one of the most sensitive and reproducible plate-based technologies available. The assay you can set up is rapid, simple to perform, and easy to automate.

ELISA Technique Applications

The ELISA test is a plate-based rapid test used for detecting and quantifying proteins, peptides, antibodies, and hormones in a liquid sample.

It may be used to diagnose:

  • HIV
  • Lyme disease
  • Pernicious anemia
  • Rocky Mountain spotted fever
  • Rotavirus
  • Squamous cell carcinoma
  • Syphilis
  • Toxoplasmosis
  • Varicella-zoster virus
  • Zika virus

ELISA Assay test includes 4 different steps:

  1. The solid phase is performed on a 96-well polystyrene plate. The function of the solid phase is to immobilize a specific lipoprotein target, which is passively adsorbed. This step is called coating.
  2. The bound target is complexed with an excess of a specific anti-target linked to an enzyme, called conjugate.
  3. After an incubation step, the plate is washed to eliminate any excess of unbound conjugate in the reaction medium.
  4. Finally, the enzyme activity of the bound enzyme 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 lipoprotein target-antibody combination, the assay is called:

  • Direct ELISA
  • Indirect ELISA
  • Competitive ELISA
  • Sandwich ELISA

Direct ELISA

The target lipoprotein (antigen in the picture) is bound to the bottom of the microplate well and is recognized by a specific enzyme-conjugated antibody that allows detection through the Chromogen/Substrate reaction.

Indirect ELISA

The target lipoprotein (antigen in the picture) is bound to the bottom of the microplate well, then an antigen-specific antibody (primary antibody in the picture) is added. A secondary enzyme-conjugated antibody that binds to the first antibody is added, allowing its detection through the Chromogen/Substrate reaction.

Competitive ELISA

The target lipoprotein (antigen in the picture) is bound to the bottom of the microplate well.

The sample plus an antigen-specific enzyme-conjugated antibody (primary antibody conjugate in the picture) are added to the wells. If the sample includes an antigen (inhibitor antigen in the picture), it competes with the antigen bound to the well for binding to the specific antibody. Unbound material is washed away. The more antigen is in the sample, the less specific antibody ends up being bound to the coated antigen, and the lower is the final signal.

The Chromogen/Substrate reaction allows detecting the specific enzyme-conjugated antibody bound to the antigen.

Sandwich ELISA

For this type of ELISA, two antibodies specific to two different epitopes on the target lipoprotein (antigen in the picture) are used. The capture antibody (primary antibody in the picture) is bound to the bottom of the microplate well and binds to one antigen epitope. The detection antibody (secondary antibody in the picture) binds to a different antigen epitope and is conjugated to an enzyme, allowing detection through the Chromogen/Substrate reaction.

An example of ELISA application using a 96-well plate

The following picture shows a plate at the end of the assay. An HRP conjugate has been used with TMB + H2O2 as chromogen/substrate.

The yellow color indicates that the target protein is present. The higher degree of the color, the higher concentration of the target protein.


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Image source: Molecular Devices