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Direct detection and Indirect detection
ELISAs, by definition, take advantage of an enzymatic label to produce a detectible signal that is directly correlated to the binding of antibody to an antigen. There are a few different types of enzymes and enzyme substrates that are typically used for ELISAs and a few slightly different methods for incorporating the enzyme step into the process. The final assay signal is measured with a spectrophotometric or fluorescent plate reader (depending upon the substrate chosen).
One aspect of ELISA terminology that often leads to confusion is the variability in the way the terms direct and indirect are applied. We will adhere to the use of these terms as they apply to the detection portion of the assay as indicated below:
Antibodies are directly labeled with alkaline phosphatase (AP) or horseradish peroxidase (HRP); this is the most common ELISA detection strategy. HRP and AP substrates typically produce a colorimetric output that is read by a spectophotometer. Detection can also occur by fluorescently-labeled antibodies [here the assay is usually termed a fluorescence-linked immunosorbent assay (FLISA)]
Antibodies are coupled to biotin, followed by a streptavidin-conjugated enzyme step; this is most common.
Additionally, it is possible to use unlabeled primary antibodies followed by enzyme-coupled or biotinylated secondary antibodies. If the secondary antibody is biotinylated, then a tertiary step is required for detection. In this case treatment with the streptavidin-enzyme conjugate, followed by an appropriate substrate.
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