Posted by kansasbio
In performing an ELISA, enzymes are employed as a label that will produce the ability to detect a signal that indicates an antibody has bound to a specific antigen. The process can use various different types of enzymes and enzyme substrates, with slightly differing methods of incorporating them. Depending on the substrate selected, either a florescent or spectrophotometric plate reader can measure the signal to achieve the final assay.
The two ELISA detection options, indirect and direct, largely determine the sensitivity of an ELISA measurement. However, the terms for the strategies can be confusing because they can have various applications. For our purposes, the terms, when applied to the detection portion of the ELISA, will be defined as follows:
Direct detection refers to the most common strategy of this type in use. This involves directly labeling antibodies using horseradish peroxidase (HRP) or alkaline phosphatase (AP) substrate. These will produce a color reagent that will allow colorimetric measurement by a spectrophotometer. A fluorescence-linked immunosorbent assay also can be attained using antibodies that are fluorescent-labeled.
Direct detection is quick, needing fewer steps to achieve. In addition, cross reaction of a secondary antibody is eliminated. However, the process allows only minimal amplification of the signal. It also can be time consuming when each specific ELISA system requires the labeling of primary antibodies.
Indirect detection of the most common type involves the coupling of antibodies to biotin, which is followed up by applying a streptavidin-conjugated enzyme. The use of primary antibodies that are unlabeled also is possible when followed with biotinylated or enzyme-coupled secondary antibodies. Another step is needed for detection, however, if a biotinylated secondary antibody is used, requiring in this case a streptavidin-conjugate treatment with a suitable substrate.
Indirect detection is the most often used format for ELISA. It affords the use of an extensive variety of labeled secondary antibodies that are available on the commercial market. It also offers versatility, since that same labeled antibody can be used for detection with many primary antibodies that are made in a single species. Signal amplification can readily be achieved, so sensitivity is enhanced. However, there is the chance for cross-reactivity with the secondary antibody, which can create a non-specific signal. The process also requires an additional incubation step.
This entry was posted in Elisa.