E E E 111

Immunoglobulin or cytokine

I Enzyme- linked

Immunoglobulin or cytokine

■ Antigen or capl ure anl foody

I Enzyme- linked

O Enzyme stibstrate

© Colored product

Figure 1 The ELISPOT assay. (A) Added cells secrete immunoglobulin or cytokine which binds locally to antigen or is captured by antibody bound to the plastic or nitrocellulose solid phase. (B) Cells are removed. Locally secreted product Is detected by enzyme-linked antibody specific for immunoglobulin or cytokine. (C) The site of immunoglobulin or cytokine secretion is revealed by addition of enzyme substrate that yields an Insoluble colored product. (D) Colored spots represent the secreted product of a single cell and can be counted by eye or under low magnification.

Figure 1 may be altered. For example, FLISPOT assays have now been performed using biotin-labeled antibody together with avidin-labeled enzyme. A further refinement is the use of antibody/enzyme/ substrate combinations that enable detection of two distinct Ig isotypes simultaneously, by virtue of different colored substrate products.

One of the most important aspects to optimize in the ELISPOT assay is the solid phase which consists of the combination of vessel surface and coated antigen or antibody. Relatively high levels of some antigens, for example ovalbumin, are required when coating plastic surfaces while other molecules such as purified Ig effectively coat the plastic surface at concentrations 10-20-fold less than is required for ovalbumin. Capture antibody (i.e. coated Ig) is therefore a useful alternative and is mostly used when one wishes to detect ISCs of no particular antigenic specificity (total ISCs) or for the detection and enumeration of cells secreting factors such as cytokines.

For the detection of antigen-specific ISCs where antigen is only available in small amounts (for example, purified viruses) or does not bind readily to plastic, other modifications have been introduced. The first is the use of nitrocellulose rather than plastic in the floor of the assay vessel. Nitrocellulose has a much greater capacity to bind protein than plastic and so the efficiency of antigen binding is substantially increased. A second modification is the labeling of the antigen in such a way that binding to the plastic surface is unnecessary. For example, the plastic surface can be coated with avidin and biotinylated antigen added. Alternatively, it is possible to use antigen directly coupled to an enzyme. The plastic-surface is coated with an isotype-specific capture antibody which binds all antibody of a particular iso-type secreted by the added ISCs. Antigen specificity is achieved by adding the enzyme-coupled antigen which is bound only at the localized sites where antigen-specific immunoglobulin was secreted and captured.

The range of specificities of ISCs now detected in both experimental animal systems as well as in humans is considerable and varies from commonly used antigens such as ovalbumin and haptens to viruses and insulin and molecules of importance in autoimmunity such as thyroglobulin, myelin basic protein and rheumatoid factor. ISCs specific for particulate antigens including platelets, sheep erythrocytes and whole bacteria as well as purified bacterial antigens such as polysaccharide have also been enumerated. There is no apparent limitation on the antibody isotype that can be detected and the discriminating power of the ELISPOT assay means that ISCs present at very low frequency (such as IgE-ISCs) can now be examined readily.

Successful detection of cells secreting a range of cytokines has been achieved, and includes those producing interleukin la (IL-la), IL-1 (3, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, interferon y (IFNy) and tumor necrosis factor (TNF). Direct comparisons between ELISA and ELISPOT assays for cytokine production by T cell clones in vitro has indicated that the ELISPOT assay for CSCs is between 10 and 200 times more sensitive than cytokine detection by ELISA performed on culture supernatant. CSCs directly ex vivo as well as after in vitro stimulation have been used successfully in the ELISPOT assay attesting to the versatility and utility of the technique.

In conclusion, the ELISPOT assay is a simple, versatile yet powerful technique for the detection and enumeration of cells liberating Ig or cytokine. The technique can be modified in a variety of ways to suit individual needs and circumstances and to optimize sensitivity. Its application to the detection of cells secreting molecules other than Ig or cytokine should also be considered. The detection of bone marrow-derived mast cells secreting mast cell protease II, is one published example of an alternative application for the ELISPOT assay.

See also: Antibodies, detection of; Cell-mediated immunity; Cytokine assays; Cytokines; Enzyme-linked immunosorbent assay (ELISA); Humoral immunity; Immunoassays; Plaque-forming cell (PFC) assays.

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