Enzyme‐Linked Immunosorbent Assay


Enzyme‐linked immunosorbent assay (ELISA) is a highly sensitive immunoassay utilising enzyme‐conjugated antibodies, with antigen or antibodies bound to a solid support. The assay measures changes in enzyme activities proportional to the antigen or antibody concentrations involved in the underlying immune reactions. The assay exists in many variants and can be designed both for detection of antigen‐specific antibodies and for quantitative measurements of virtually any substance. Major applications include diagnostic assessment of disease‐related antibodies and detection and quantitation of hormones, cytokines and antigens from common pathogens. A special variant of the assay, ELISpot, can be used for studying secretion of analytes by single cells and is widely used for studying immune responses to vaccination and natural infection at the level of individual T and B cells.

Key Concepts

  • ELISA assays can be configured in many ways and used in many fields of science.
  • The combination of high sensitivity and specificity, simple procedure and the possibility to use both for analysing individual samples and for automated high‐throughput screening makes ELISA an ideal assay for many purposes.
  • Indirect ELISA is the basis for a large number of serological assays where detection of antibodies to infectious pathogens, allergens and autoimmune target antigens is the main diagnostic indicator of disease.
  • Sandwich ELISA, using an immobilised capture antibody and a labelled detection antibody, allows highly specific and sensitive detection and quantitation of virtually any antigenic substance including hormones, cytokines, toxins and microbial pathogens.
  • ELISpot allows detection of secreted products at the single‐cell level, making it possible to identify individual antigen‐specific T and B cells based on their secretion of cytokines and antibodies, respectively.

Keywords: ELISA; ELISPOT; immunoassay; antibody; antigen; serology; diagnostic

Figure 1. Schematic outline of a sandwich or capture ELISA.
Figure 2. ELISpot showing T cells responding with Interferon‐γ secretion after 18 h of exposure in vitro to antigenic peptides from cytomegalovirus, CMV (a). Each spot indicates a responding cell and a control well (b) that without addition of antigen shows no or few secreting cells.


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Further Reading

Butler JE (1994) Enzyme linked immunosorbent assay. In: van Oss CJ and van Regenmortel MHV (eds) Immunochemistry, pp. 759–803. New York: Marcel Dekker.

Calarota SA and Baldanti F (2013) Enumeration and characterization of human memory T cells by enzyme‐linked immunospot assays. Clinical and Developmental Immunology, 2013, Article ID 637649.

Hornbeck P (1991) Enzyme‐linked immunosorbent assay. In: Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM and Strober W (eds) Current Protocols in Immunology, pp. 2.1.1–2.1.22. New York: Greene Publishing Associates & Wiley Interscience.

Klinman DM and Nutman TB (1994) ELISPOT assay to detect cytokine secreting murine and human cells. In: Coligan JE, Kruisbeek AM, Margulies DH, Shevach EM and Strober W (eds) Current Protocols in Immunology, pp. 6.19.1–6.19.8. New York: Greene Publishing Associates & Wiley.

Nielsen UB and Geierstanger BH (2004) Multiplexed sandwich assays in microarray format. Journal of Immunological Methods 290: 107–120.

Stott DI (1994) Immunoblotting, dot‐blotting, and ELISPOT assays: methods and application. In: van Oss CJ and van Regenmortel MHV (eds) Immunochemistry, pp. 925–948. New York: Marcel Dekker.

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Paulie, Staffan, and Perlmann, Hedvig(Jan 2016) Enzyme‐Linked Immunosorbent Assay. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002625.pub3]