ELISA Assay Technology

Applications & Techniques

The ELISA assay (enzyme-linked immunosorbent), also called EIA (enzyme immunoassay), 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. ELISA 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.


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, as an example, the following disease states:

  • Hepatitis
  • Retrovirus
  • ToRCH (Toxoplasmosis, Rubella, Cytomegalovirus, Herpex Simples)
  • EBV
  • MMVZ (Measles, Mumps, Varicella, Zooster)
  • Syphilis
  • Tubercolosis
  • Autoimmunity

ELISA assay test consists of 4 different main phases:

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

  • direct ELISA
  • indirect ELISA
  • competitive ELISA
  • sandwich ELISA

Direct ELISA assay

The target lipo-protein (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, by the Chromogen/Substrate reaction.

Indirect ELISA assay

The target lipo-protein (antigen in the picture) is bound to the bottom of the microplate well,then a specific antibody to the antigen (primary antibody in the picture) is added. Secondary enzyme conjugated antibody that binds to the first antibody is added, allowing its detection, by Chromogen/Substrate reaction.

Competitive ELISA assay

The target lipo-protein (antigen in the picture) is bound to the bottom of the microplate well.
Sample plus specific enzyme conjugated antibody (primary antibody conjugate in the picture) to the antigen are added to the wells. If there is an antigen in the sample (inhibitor antigen in the picture), it competes with the antigen bound to the well for binding the specific antibody. Unbound material is washed away. The more antigen is in the sample, the less specific antibody ends up bound to the coated antigen, and the lower the final signal.
The Chromogen/Substrate reaction allows the final detection of the specific enzyme conjugated antibody bound to the antigen.

Sandwich ELISA assay

For this type of ELISA two antibodies specific to two different epitopes on the lipo-protein target (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 one epitope of the antigen. The detection antibody (secondary antibody in the picture) binds the antigen at a different epitope and is conjugated to an enzyme, allowing detection, by the Chromogen/Substrate reaction.

ELISA assay: An example of an assay using a 96-well plate

The following picture shows, as example, a plate at the end of the assay. In 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.

Discover Biomat’s most requested ELISA 96 Well plates:

Check out Biomat’s ELISA Plates for ELISA Assay Kit.

Image source: Molecular Devices