Introduction to ELISA

Enzyme Linked Immunosorbent Assay (ELISA) is a powerful technique for detection and quantitation of biological substances such as protein, peptides, antibodies, and hormones. By combining the specificity of antibodies with the sensitivity of simple enzyme assay, ELISA can provide a quick and useful measurement of the concentration of an unknown antigen or antibody.

 

Currently, there are three major types of ELISA assays commonly used by researchers. They are: indirect ELISA, typically used for screening antibodies; sandwich ELISA (or antigen capture), for analysis of antigen present; and competitive ELISA, for antigen specificity. PBL's IFN ELISA kits use the sandwich strategy.

 

The "sandwich" technique is so called because the antigen being assayed is held between two different antibodies each recognizing particular epitopes on the antigen. Because of both antibodies are specific for the target protein, this method can increase the assay specificity two to five times over other direct or indirect detection methods. In this method:

 

  1. Plate is coated with a capture antibody.
  2. Sample is then added, and any antigen present binds to capture antibody.
  3. The detecting antibody is then added and binds to a different region (epitope) of the antigen.
  4. Enzyme linked secondary antibody is added and binds to the detecting antibody.
  5. The substrate is then added and the reaction between the substrate and the enzyme produces a color change. The optical density (OD) values can be measured spectrophotometrically.

Sandwich ELISA diagram

 

Optimizing an ELISA assay not only requires time, but it also requires the careful selection of antibodies and enzyme-substrate reporting system. However, once it has been optimized, sandwich ELISA technique is fast and accurate. If a purified antigen standard is available, this method can be used to detect the presence and to determine the quantitative amount of antigen in an unknown sample. The sensitivity of the sandwich ELISA is dependent on 3 factors:

 

  1. The number of molecules if the first antibody that are bound to the solid phase, namely, the microtiter plate.
  2. The avidity of the antibodies (both capture and detection) for the antigen
  3. The specific activity of the detection antibody that is dependent on the number and type of labeled moieties it contains.

It is important to note that while an ELISA assay is a useful tool to detect the presence and the quantity of an antigen in the sample, it does not provide information concerning the biological activity of the sample.  It cannot be used to discriminate active or non-active forms of a protein, and it can also detect degraded proteins that have intact epitopes.

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