Chromogenic immunohistochemistry: The cell is exposed to a primary antibody (red) that binds to a specific antigen (purple square). The primary antibody binds a secondary (green) antibody that is chemically coupled to an enzyme (blue). The enzyme changes the color of the substrate to a more pigmented one (brown star). |
Reporters / Chromogenic / Fluorophore
Traditional ELISA typically involves chromogenic reporters and substrates that produce some kind of observable color change to indicate the presence of antigen or analyte. Newer ELISA-like techniques use fluorogenic, electrochemiluminescent, and quantitaoppositiontive PCR reporters
to create quantifiable signals. These new reporters can have various advantages, including higher sensitivities and multiplexing.[13][14] In technical terms, newer assays of this type are not strictly ELISAs, as they are not "enzyme-linked", but are instead
linked to some nonenzymatic reporter. However, given that the general principles in these assays are largely similar, they are often grouped in the same category as ELISAs.
Reporter molecules vary based on the nature of the detection method, the most popular being chromogenic and fluorescence detection mediated by an enzyme or a fluorophore, respectively. With chromogenic reporters, an enzyme label reacts with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope. While the list of enzyme substrates is extensive, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes used
most extensively as labels for protein detection. An array of chromogenic, fluorogenic and chemiluminescent substrates is available for use with either enzyme, including DAB or BCIP/NBT, which produce a brown or purple staining, respectively, wherever the enzymes are bound. Reaction with DAB can be enhanced using nickel,[7] producing
a deep purple/black staining.
Fluorescent reporters are small, organic molecules used for IHC detection and traditionally include FITC, TRITC and AMCA, while commercial derivatives, including the Alexa Fluors and Dylight Fluors, show similar enhanced performance but vary in price. For chromogenic and fluorescent detection methods, densitometric analysis of the signal can provide semi- and fully quantitative data, respectively, to correlate the level of reporter signal to the level of protein expression or localization.
https://en.wikipedia.org/wiki/Immunohistochemistry#IHC_reporters |