ELISABased Assays

The enzyme-linked immunosorbent assay (ELISA) is one of best-established and most often-used screening technologies since the start of the HTS era. Today, ELISA technology is widely used in the field of diagnostics, and can be easily transferred to a microtiter format. Although a variety of different assay protocols exist, all of them depend upon the availability of an antibody with a specificity for the antigen of interest. Binding of the primary antibody to the antigen can be detected by a secondary antibody which recognizes species-specific sequence differences in the FC portion of the primary antibody. These secondary antibodies are often coupled with an enzyme such as alkaline phosphatase or horseradish peroxidase. When provided with appropriate substrates, these enzymes can produce colored, chemifluorescent or chemiluminescent products, which can easily be quantified [42]. The amount of colored product is, therefore, within certain limits, proportional to the amount of antigen in the well.

In combination with cellular screens, ELISA-type technologies can be used, for example, to detect and measure antigens produced by the cells. Examples of such applications are the detection of cell-surface [43] or viral antigens [44]. A novel variant of ELISA technology is the antibody-mediated quantification of phosphorylated kinase substrates [45]. These Phosphospecific Antibody Cell-based PLISA (PACE) screens are used to monitor the activity of kinase pathways within cells, which is of considerable value as kinases have become a major focus of drug discovery programs.

As an example, Versteeg et al. have developed an ELISA test to detect p42/ p44 MAP kinase activation, p38 mitogen-activated protein kinase (MAPK), phosphokinase B and cAMP-response-element-binding (CREB) protein [46]. A PACE-based screening not only allows for large-scale analysis of signal transduction within a cellular environment, but also determines if a compound can sufficiently penetrate the cell membrane. In some PACE applications the secondary antibody used can be labeled with a fluorophore, which allows localization of the signal specifically to cells, or even to subcellular compartments.

ELISAs are typical examples of nonhomogeneous assay technologies, and the many washing steps require huge amounts of washing buffers. Therefore, they are not favored by most HTS laboratories as the many necessary steps slow down the assay protocol and reduce throughput. Despite the difficult screening logistics, however, ELISA technologies are well established and provide robust and sensitive measurements, and homogeneous variants of the detection technology have been described [45].

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