ELISA Assay Technology

Applications & Techniques

ELISA Assay Technology is a versatile immunoassay technique that falls into the category of immunoenzymatic tests; it is ideal for detecting and quantifying a specific molecule in a complex sample.

The ELISA Assay (Enzyme-Linked Immunosorbent Assay), 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. 

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 technique 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

ELISA applications may be used, for example, to diagnose:

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

The 4 different main phases of ELISA Assay Technology

  1. The solid phase consists of a 96-well polystyrene plate, although other materials can be used. 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 enzymatic 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 ELISA Assay Technology is called:

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


Direct ELISA

In the Direct ELISA test plate, 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

In the Indirect ELISA test plate, 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. A secondary enzyme conjugated antibody that binds to the first antibody is added, allowing its detection, by Chromogen/Substrate reaction.

Competitive ELISA

For the Competitive application of ELISA technique, 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

For this type of ELISA Assay 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.

An example of ELISA application using a 96-well plate

The following picture shows, as example, a plate at the end of the ELISA 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 and their technical guide:


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Image source: Molecular Devices