Antibody Detection in IHC by Chromogenic Substrates

Immunohistochemistry (IHC) is a method of detecting antigens in tissues or cells using antibodies to bind with them, and then visualizing the antibodies through chromogenic or fluorescent techniques. Chromogenic techniques use enzymes to change soluble substrates added to the sample after antigen-antibody binding, into insoluble products that give a color to the target antigens.

The enzymes used in these methods are generally conjugated to another antibody that is raised against the primary antibody (the one that recognizes and binds to the antigen itself), known as the secondary antibody. Sometimes, a primary antibody is directly conjugated to the enzyme, avoiding the need for a secondary antibody.

The enzymes that are used most widely include horseradish peroxidase (HRP), which acts on the substrate 3,3'-diaminobenzidine (DAB) to convert it into a brown product; and alkaline phosphatase (AP) which acts on 3-amino-9-ethylcarbazole (AEC) to produce a red-colored product.

Chromogenic detection methods have greater sensitivity than fluorescence techniques because the signal is amplified more intensely. The colored products are often stable to light, which means the prepared slides can be kept in storage for years for review and to build up a database.

A third advantage is that there is no need for specialized light filters and light sources, as it uses a standard microscope. On the other hand, the protocol for this method is more drawn out, including additional blocking and incubation steps, compared to fluorescent detection methods.

There are four primary methods by which indirect chromogenic detection is carried out. Some are biotin-based, such as the avidin-biotin complex (ABC) or a labeled streptavidin-biotin binding (LSAB) complex. Others include the use of a polymer or micropolymer complex.

The Avidin-Biotin Complex (ABC) Method

Originally, the ABC technique used secondary antibodies conjugated to biotin with an avidin-biotin- reporter enzyme complex to produce a high-intensity signal.

Labeled Streptavidin-Biotin (LSAB) Method

Current chromogenic detection depends on the use of streptavidin rather than avidin, and is known as the LSAB variant of ABC. This reduces non-specific binding in the tissues because of the absence of glycosylation in streptavidin, which also possesses an isoelectric point which is more neutral than avidin.

Polymer Methods

The biggest problem with the biotin-based techniques is the background staining that results from the presence of endogenous biotin in tissues such as the brain. While its level is decreased following fixation with formalin and paraffin embedding, biotin can become exposed after antigen retrieval.

Endogenous biotin is also an issue with frozen sections. This can be tackled in two ways: either more steps using biotin blocking solutions can be added to minimize background signals or polymer-based methods may be used instead. The original polymer-based techniques made use of a dextran spine to attach multiple enzymes and secondary antibodies to it.

The later micropolymer or compact polymer methods are better, as they are associated with fewer aggregates, since the detection complex is smaller. They are therefore more sensitive, since the degree to which the tissue is penetrated is better and the background staining due to endogenous biotin is lower.

Multicolor Detection IHC

If the aim of the test is to detect more than one antigen, chromogens which give different colors may be used to stain up to three antigens at once. For this kind of testing, primary antibodies must come from different species, or the antigen to which the primary antibody (directly conjugated with the enzyme which acts on the chromogenic substrate) must be quite abundant.

Another option is the use of blocking agents to allow one primary antibody isolated from one host species to be used for staining. After this, the sites on the primary antibody to which the secondary antibody binds are blocked. Finally, another primary antibody from the same species is used to stain another antigen.

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: Apr 1, 2019 at 6:04 AM

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