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تسجيل

Enzyme Immunosorbent Assays

المؤلف:  Wilson, K., Hofmann, A., Walker, J. M., & Clokie, S. (Eds.)

المصدر:  Wilson and Walkers Principles and Techniques of Biochemistry and Molecular Biology

الجزء والصفحة:  8th E , P272-273

2026-05-06

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The vast majority of immunoassays carried out today fall into the category of enzyme linked immunosorbent assays ( ELISAs). These are routinely used as diagnostic methods that detect antigens of infectious agents or antibodies against them in bodily fluid. In this technique, the antigen or antibody from the sample tested is immobilised from the liquid phase (plasma or serum) onto a solid phase, typically wells of a microtitre plate. Immobilisation is achieved either directly, or indirectly by the use of a coating antibody (also called capture antibody), which actively traps antigen in the solid phase. The immobilised antigen will bind specific antibodies; detection of specifically bound antibody or of the antigen itself is thus achieved with another antibody, which is labelled with a reporter enzyme producing a signal (usually a colour change) when the enzyme substrate is added to the antibody–antigen–enzyme complex ( Figure 1).

Fig1. Schematic diagram of the sandwich ELISA.

There are many variations of this method, but all of them rely on the formation of the antibody–antigen complex and its detection by the reaction of the reporter enzyme. Since these assays rely on a stepwise addition of layers, with each one being linked to the one before, they are collectively termed sandwich ELISA . In the first layer, the coating antibody irreversibly binds the antigen; in this process, the available antigen is concentrated until saturation has been reached. This is particularly useful when testing for low levels of antigens in fluids such as blood serum. After the antigen has been applied and prior to addition of the detecting antibody, plate blocking may be done by addition of a protein that does not specifically interfere with the ELISA (e.g. casein from milk powder or bovine serum albumin). Depending on how many detection layers are employed, enzyme immunosorbent assays can be classified into direct (e.g. double anti body sandwich) and indirect (e.g. triple antibody sandwich) ELISAs (see also Table1).

Table1. Comparison of direct and indirect ELISA