ELISA: Detection Options - Direct and Indirect


ELISAs, by definition, take advantage of an enzymatic label to produce a detectible signal that is directly correlated to the binding of an antibody to an antigen. A few different types of enzymes and enzyme substrates are typically used for ELISAs. Several methods for incorporating the enzyme step into the process can also be applied. 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:

Direct ELISA detection

Antibodies are directly labeled with alkaline phosphatase (AP) or HRP; this is the most common ELISA detection strategy. HRP and AP substrates typically produce a colorimetric output that is read by a spectrophotometer. Detection can also occur by fluorescently-labeled antibodies; here the assay is usually termed a fluorescence-linked immunosorbent assay (FLISA).

Indirect ELISA detection

Antibodies are coupled to biotin, followed by a streptavidin-conjugated enzyme step. Alternatively, 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 is followed by an appropriate substrate.