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Double Antibody Sandwich ELISA (DAS ELISA)

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 , P274

2026-05-06

268

 Double antibody sandwich ( DAS) ELISA (also called direct ELISA) is probably the most widely used immunochemical technique in diagnostics. The principle is that the anti gen is immobilised on a solid phase by a primary coating antibody and detected with a second antibody that has been labelled with a marker enzyme (Figure 1). The anti gen creates a bridge between the two antibodies and the presence of the enzyme causes a colour change in the chromogenic (colour-producing) substrate. The marker enzyme used is usually either horseradish peroxidase or alkaline phosphatase. Other enzymes have been reported to give higher sensitivity, but this is at the expense of more complex substrates and buffers. In some systems, the enzyme is replaced with a radioactive label and this format is known as the immunoradiometric assay (IRMA). DAS ELISA is used extensively in horticulture and agriculture to ensure that plant material is free of virus. Potato tubers, for example, that are to be used as seed for growing new crops have to be free of potato viruses, and screening in particular for potato leafroll virus (PLRV) is carried out by DAS ELISA. PLRV antibodies are coated onto the wells of ELISA plates and then the sap to be tested is added. After incubation, the plates are washed and PLRV antibody conjugated to alkaline phosphatase is added. After further incubation and washing, the substrate is used to identify the positive wells, caused by the presence of the antigen (PLRV) in the sandwich of antibodies.

Fig1. Schematic diagram of the double antibody sandwich (DAS) ELISA.