Enzyme Linked-Immunosorbent Assay (ELISA)

the four steps of an enzyme-linked immunosorbent assay (ELISA)

Overview & Resources

ELISA, short for enzyme-linked immunosorbent assay, is a plate-based assay technique used in analytical biochemistry and other fields of study to detect and quantify soluble substances, such as proteins, antibodies, and peptides in complex mixtures. This technique is also sometimes referred to as EIA, or enzyme immunoassay, and although the naming differs, the technology is the same.

ELISA was originally described by Engvall and Perlmann in 1971.  ELISA enables the analysis of protein and other samples, which have been immobilized in microplate wells using specific antibodies. These immunoassays are typically performed in 96-well or 384-well polystyrene plates that passively bind antibodies and proteins. 

The assays are simple to design and easy to perform. Using reagents to bind and immobilize the proteins to the microplate surface makes the separation of bound and unbound material during the assay much simpler than other separation methods.

Furthermore, using high-affinity antibodies to wash away non-specific bound materials makes ELISA a powerful tool for measuring specific analytes within a crude preparation.

While there are numerous ELISA variants used for different situations, each method depends on the same basic elements:

  1. Coating and capture: Antigens are either directly or indirectly immobilized to the surface of polystyrene microplate wells.
  2. Plate blocking: The addition of an irrelevant protein or other molecule covers all unsaturated surface-binding sites of the microplate wells.
  3. Probing & detection: Antigen-specific antibodies are incubated and bind to the antigens based on affinity.
  4. Signal detection: Lastly, the signal generated is measured via the direct or secondary tag on the specific antibody.

Some of the most commonly used enzyme labels in ELISA include horseradish peroxidase (HRP) and alkaline phosphatase (AP). There are other enzymes used as well, however, including β-galactosidase, acetylcholinesterase, and catalase. Substrates are also employed in the immunoassay.

The choice of substrate depends on the assay sensitivity and the type of instrument being used to detect a signal, such as a fluorometer, luminometer, or spectrophotometer. Further, the various selections available have all been optimized for use in ELISA with an HRP or AP conjugate.

ELISA Resources

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