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What are the different detection methods for IHC?



Courtesy of Dr Renaud Burrer (Histalim, Montpellier, France)
Immunohistochemistry (IHC) is a powerful and routinely used technique for the detection, localization, and scoring of cellular macromolecules in preserved tissues. Independent of the specific method used, the very first step of this technique is the selective binding of the primary antibody with its specific target. This interaction can then be visualized, directly or indirectly, with a chromogenic reaction catalyzed by reporter enzymes such as Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP). In the case of a direct detection, the primary antibody (AbI) itself is labeled with HRP or AP enzymes (Fig. 1A). However, the preferred strategy is often an indirect detection, where the unlabeled primary antibody is recognized by a labeled secondary antibody (AbII; Fig. 1B).

direct and indirect IHC detection

Figure 1. Schematic representation of direct (A) and indirect (B) IHC detection. S: Chromogenic substrate. P: Colored insoluble product. Reporter enzyme is indicated by the red star.


In both cases, a chromogenic substrate is eventually converted in a colored precipitate by the reporter enzyme, staining the tissue in correspondence to the targeted antigen. The indirect detection method is commonly used because it ensures an amplified signal and higher sensitivity, as several secondary antibodies can bind different epitopes of the primary antibody. In addition, it also allows higher flexibility as just few types of secondary antibodies can be used to detect a wide range of primary antibodies against the different antigens. A few of the most commonly used detection methods are described below.


Peroxidase/Anti-Peroxidase (PAP) Detection Method

Pioneered by Sternberger in the seventies, PAP is based on the formation of a peroxidase/anti-peroxidase complex, connected to the primary antibody via a secondary “bridge” antibody. Exploiting the bivalent properties of IgG, the PAP complex contains three peroxidase molecules and two anti-peroxidase antibodies (Fig. 2). Compared to a simple indirect detection (reporter enzyme directly linked to the secondary antibody), this method allows for higher sensitivity because several enzyme molecules are localized per antigenic site. In addition, since the enzyme is not chemically modified, there is low risk of reducing its apparent activity.

Peroxidase detection method for IHC

Figure 2. Schematic representation of the Peroxidase/Anti-Peroxidase (PAP) detection method. Reporter enzyme is indicated by the red star.



ABC (Avidin-Biotin Complex) Detection Method

This popular method is based on the almost irreversible non-covalent interaction between biotin and avidin. Avidin is a large tetrameric glycoprotein (67-68 kDa) found in the egg-whites of amphibians, birds, and reptiles. Each subunit can bind a biotin molecule with extremely high affinity so that up to four biotins can bind to the same avidin molecule. Biotin is a small vitamin (~244 Da, also referred to as vitamin H or B7) acting as an essential co-enzyme for mammalian carboxylases in gluconeogenesis, fatty acids synthesis, and amino acids metabolism. It is ubiquitous in mammals’ tissues in little amounts, with a slightly higher concentration in liver. Thanks to its moderate size, it can be conjugated to other macromolecules without altering its activity.

The ABC method is a three-step detection method: the primary antibody binds to the antigen in the tissue; it is in turn recognized by the biotinylated secondary antibody; complexes formed by avidin and biotinylated enzyme (ABC) can then attach to the latter. Since the avidin tetramer can bind four biotinylated biotins, large ABC lattices containing several reporter enzymes are formed, leading to a strong amplification of the signal at the antigenic site (Fig. 3).

The elevated enzyme:antibody ratio and the consequent high sensitivity is the main advantage of this procedure, which is still one of the most widely used both in the research and in the diagnostic field. However, a few drawbacks need to be considered. For example, it can be difficult for the large ABC structures to diffuse efficiently in the tissue. Moreover, the presence of endogenous biotin is a potential source of background. With its high isoelectric point (pI=10), the avidin is positively charged and can bind negatively charged molecules (e.g. nucleic acids). In addition, its carbohydrate moiety can interact with other molecules such as lectins. These avidin-related issues can be overcome thanks to the LSAB method described below.

ABC detection method for IHC

Figure 3. Schematic representation of the ABC detection method.



Labeled Streptavidin Biotin (LSAB) Detection Method

This technique can be considered an evolution of the previous one, as the main difference is that it uses streptavidin instead of avidin. Streptavidin is a tetrameric protein isolated from the bacterium Streptomyces avidinii. It has little sequence homology with avidin but their quaternary structures are very similar. Streptavidin can, in fact, bind four biotin molecules with high affinity as well, but it is not glycosylated (thereby eliminating non-specific interactions with lectin-like molecules) and it has a neutral pI (thereby limiting the risk of electrostatic binding). Thus, the LSAB method reduces background staining issues and it is ten times more sensitive than the ABC one. Similar to the ABC strategy, this is a three-step procedure: biotinylated AbII recognizes the AbI, but in this case, the streptavidin is directly conjugated to the reporter enzyme.



Figure 4 depicts IHC staining obtained with SAVIEW® PLUS detection solutions from Enzo Life Sciences. These kits are based on an improved version of the LSAB method: streptavidin molecules are labeled with a nanopolymer of the reporter enzyme thanks to a proprietary technology from Enzo’s laboratories. The SAVIEW® ready-to-use reagents enables a sensitive, enhanced streptavidin-based detection, with the highest flexibility. Choose between SAVIEW PLUS® HRP- or AP- labeled reagents, select the chromogen you prefer and get a sharp strong staining of your tissue with a manual protocol or with an automated platform.



Figure 4. IHC staining obtained with SAVIEW® PLUS detection solutions. A. Cervical tissue stained with anti-Ki-67 antibody (mouse), then biotinylated anti-mouse secondary antibody, and followed by SAVIEW® PLUS HRP reagent (ENZ-ACC102). B. CD44 staining on human section obtained with SAVIEW® PLUS AP (ENZ-ACC111) and HIGHDEF® Green AP Chromogen/Substrate (ENZ-ACC130).



Polymer-based Detection Method

Despite the sensitivity improvement, there is still a concern of potential false positives related to the presence of endogenous biotin with the LSAB detection method. Specific blocking procedures do exist, but this potential issue remains especially in biotin-rich tissues (e.g. liver and kidney) or in frozen tissue sections, in which biotin is better preserved compared to paraffin-embedded samples. The need for a specific, sensitive, and possibly faster method led to the development of the polymer-based detection method, which completely circumvents biotin recognition and uses polymers to increase the enzyme:AbI ratio.

One way to exploit the polymer technology is to use large dextran backbones, conjugated with up to 20 secondary antibodies and 100 enzyme molecules as represented in Fig. 5A. This method allows for extremely sensitive detection and implies a two-step protocol, faster than the approaches described so far. One possible shortcoming is the high molecular weight of the dextran backbone, which can render the penetration of the polymer in the tissue and the interaction with some antigens (i.e. nuclear targets) difficult. The size of the polymers also reduces the enzyme density in the site of the primary antibody.

An alternative method has therefore been developed in order to get more compact antibody-enzyme complexes. This consists of polymerizing enzymes in small linear molecules and attaching these short polymers to antibodies, thus obtaining a high density of active reporters and minimizing the steric interference of the polymer (Fig. 5B). This second generation polymer-based detection technique strongly increases the sensitivity, the specificity, and the signal intensity of the IHC, keeping a user-friendly two-step protocol.

polymer-based detection methods for IHC

Figure 5. Schematic representation of the polymer-based detection methods. A. Dextran polymers conjugates to several reporter enzymes and secondary antibodies. B. Second generation polymer-based method, based on the generation of linear enzyme polymers, attached to the secondary antibody.


A similar nanopolymer-based detection technology is used for the POLYVIEW® PLUS detection solutions, developed by Enzo Life Sciences. These kits provide ready-to-use reagents, compatible with both manual and automated platforms, allowing for specific, crisp and intense staining in your IHC experiments (Fig. 6A). If you are looking for the possibility of a superior multiplex detection, the MULTIVIEW® PLUS detection systems may be your solution (Fig. 6B). These kits, combined with the broad palette of colors proposed by Enzo, can be used to detect up to four different targets on a single section, thus saving precious specimens and reducing reagent costs.

nanopolymer-based detection

Figure 6. IHC staining obtained with Enzo’s nanopolymer-based detection solutions. A. Cervical tissue stained with anti-Ki-67 antibody (rabbit), followed by POLYVIEW® PLUS HRP (anti-rabbit) (ENZ-ACC103). B. Human lung stained with anti-CK5/6 (red, ADI-950-210), anti-Napsin A (yellow, ADI-950-170), p63 (black, ADI-950-171) and TTF-1 (green, ENZ-ACC130), using a combination of Enzo’s POLYVIEW® PLUS HRP (anti-rabbit) Reagent (ENZ-ACC103) and MULTIVIEW® PLUS (mouse-HRP/rabbit-AP) IHC Kit (ENZ-KIT181). Picture is a courtesy of Dr Renaud Burrer (Histalim, Montpellier, France).



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Pros and cons for each of the aforementioned methods are summarized in the table below.

Type of Detection Advantages Disadvantages
Direct + Fast (potentially useful for rapid diagnostics) - No signal amplification: very low sensitivity
- High costs and poor flexibility: need for labeled primary antibodies for each target antigen
Indirect (simple) + Increased sensitivity compared to direct method
+ Lower costs and higher flexibility: only secondary antibody needs to be labeled
- Poor signal amplification: reduced sensitivity
PAP + Signal amplification: Increased sensitivity compared to previous methods
+ Reduced background staining
+ Allows higher dilutions of the primary antibody
+ No need for chemical conjugation of the molecules
- PAP complex needs to be from the same species of the primary antibody
- High level of endogenous peroxidases can be a limiting factor
- More time consuming compared to previous methods
- Might not be sensitive enough for the analysis of FFPE tissues
ABC + Faster procedure
+ Allows dilution of the primary antibody
+ Avidin-biotin complex can be used for several days
+ Strong signal amplification: higher sensitivity compared to previous methods
- Background/false positives: endogenous biotin; avidin-related non-specific binding
- Size of the complex could decrease efficiency of tissue penetration
LSAB + Reduced avidin-related non-specific staining issues
+ Allows dilution of the primary antibody
+ Strong signal amplification
+ LSAB complexes are more stable than ABC ones
- Potential background issues caused by endogenous biotin
Polymer-based + Two-step procedure: faster protocol
+ Reduced background and non-specific staining
+ High signal amplification: high sensitivity
- More expensive than previous methods
- Concerning dextran polymers: steric interference can make the penetration in the tissue difficult



Taking all this into account and based on the type of tissue you are working on, the antigen you are targeting, the primary antibody you are going to use, and the lab instruments available, you can select the best option for your IHC experiments.


Need more advice to obtain perfect immunostaining? Don’t miss our Immunohistochemistry Troubleshooting Guide, our 10 Tips for Successful Immunohistochemistry and our IHC E-book. Besides the aforementioned detection systems, Enzo offers a complete range of products for IHC detection ranging from antigen retrieval, to the mounting medium to prepare your slides. Browse the complete range of Enzo’s IHC products here: Enzo’s IHC platform. Check out the amazing potential of these products by reading the related application notes: IHC Collaborations at work. For all questions and concerns regarding any of our products, our Technical Support Team is here to assist.

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Enzo offers research kits, biochemicals and biologicals backed by over 40 years of expertise. As Scientists Enabling Scientists™, we realize the value in providing relevant information to our customers working in the fields of life sciences, drug development and clinical research. We are happy to provide simple but useful tips for improving daily tasks as well as the overall quality of your research.
With this in mind, here is an e-book that answers important questions such as:

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