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How to Choose the Best Secondary Antibody for Immunohistochemistry Application
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How to Choose the Best Secondary Antibody for Immunohistochemistry Application

Selection of the best secondary antibody can improve immunostaining and reduce false positive or negative staining. Here is some useful information that will help you to choose the best secondary antibody possible for your specific immunohistochemical application.

Species of Primary Antibody:

The secondary antibody should be against the species that primary antibody is raised. For example, if the primary antibody is raised in mouse, an anti-mouse secondary antibody should be used. If it is raised in rabbit, an anti-rabbit secondary antibody should be used.

Class and Subclass of Primary Antibody:

The secondary antibody should match the class or subclass of the primary antibody used. This is especially true for monoclonal antibodies. For example, if the primary antibody is mouse IgM, an anti-mouse IgM or anti-mouse IgG should be used.

If the primary antibody (monoclonal) is one of the mouse IgG subclasses (IgG1, IgG2a, IgG2b, IgG2c, IgG3), any of the anti-mouse IgG can be used. If the class and/or subclass of the primary antibody are not known, the anti-mouse IgG may be used since they recognize most of mouse IgG subtypes.

One can also use a secondary antibody specific to the IgG subclass of the primary antibody. For example, if the primary antibody is mouse IgG2a, an anti-mouse IgG2a secondary antibody can be used and this is especially useful for double labeling methods.

Polyclonal antibodies (such as rabbit, goat, sheep or donkey) are typically IgG class immunoglobulins so the anti-IgG secondary antibodies for these species may be used.

Species of Secondary Antibody:

There appears to be no evidence of species specific difference in the quality of secondary antibodies. Therefore selection of the host species should be based on other criteria.

Form of Secondary Antibody:

Affinity purified antibodies are commonly used and it gives the lowest amount of non-specific binding. However, IgG fractions may have the benefit of containing very high affinity antibodies. Therefore, they may be useful in situations where very high affinity is required (i.e. when the antigen of interest is rare or present in low abundance).

Labeled or Conjugated Secondary Antibody:

In general, secondary antibodies can be either enzyme labeled (peroxidase, alkaline phosphatase), fluorescence labeled (FITC, Alexa-Fluor, Qdot) or biotin conjugated. Peroxidase is economical, rapid and a more stable enzyme, while the alkaline phosphatase on the other hand is considered more sensitive than peroxidase particularly when colorimetric detection is used. Fluorescent labeled antibodies are commonly used for double or multiple staining methods.

The biotin-conjugated antibody is used to amplify the signal and resulting in greater sensitivity than that achieved with an enzyme or fluorescence conjugated secondary antibody alone.

Most recently several companies have developed a more sensitive detection system called polymer detection system. Among these are EnVision (DakoCytomation), ImmPRESS (Vector Labs), and MACH 2 (Biocare Medical).

Adsorbed Secondary Antibody:

Some of the secondary antibodies have been adsorbed with animal or human IgG. These antibodies are designed for particular applications to reduce non-specific background staining. For example, if working with rat tissues or cells, choose a secondary antibody that has been adsorbed with rat serum or IgG. However, such adsorbed antibodies have greatly reduced epitope recognition and may recognize some subclasses of IgG very weakly, especially those subclasses which are most closely homologous to the species they were adsorbed against. For example, do not use an anti-mouse IgG that has been adsorbed against rat IgG unless you are trying to detect a mouse primary antibody in rat tissue that contains rat immunoglobulin, or in some other tissue in the presence of a rat primary antibody. Conversely, if you wish to detect a mouse primary antibody in the absence of rat immunoglobulins, it is best to use an anti-mouse secondary antibody that has not been adsorbed against rat.

Whole IgG or F(ab')2 Fragments of the Secondary Antibody:

If working with tissues or cells that have Fc receptors (thymus, spleen, blood, hematopoietic cells, leukocytes, B cells, etc.), choose an F(ab')2fragment when possible to eliminate non-specific binding through Fc receptors present on cells. As an alternative, binding of whole molecule, secondary antibodies to Fc receptors may be blocked by incubating cells in purified IgG or normal serum from the host species of the secondary antibody.

Please note that if a primary antibody is not an F(ab')2 fragment, it may also bind to Fc receptors, and blocking with normal serum from the host species of the secondary antibody may not always be successful. In this case, using direct immunostaining method and blocking with an antibody specific to Fc receptors or normal serum (IgG) from the host species of the primary antibody may be successful.

  • Anti-IgG (H+L): This antibody reacts with both the heavy and light chains of the IgG molecule, i.e. it reacts with both the Fc and F(ab')2 portions of IgG. Anti-IgG (H+L) also reacts with other immunoglobulin classes (e.g. IgM, IgA, etc.) since all immunoglobulins share the same light chains (either kappa or lambda). This is commonly used secondary antibody.
  • Anti-IgG, Fc fragment specific: This antibody reacts with the Fc portion of the IgG heavy chain. In each case, it has been adsorbed against F(ab')2 fragments. In some cases, the antibodies are additionally adsorbed to minimize possible cross-reactivity to IgM and IgA, or to IgM alone.
  • Anti-IgG, F(ab')2 fragment specific: This antibody has been adsorbed against Fc fragments and therefore reacts only with the Fab portion of IgG. Since it reacts with light chains, it also reacts with other immunoglobulins sharing the same light chains. This antibody is used for specific applications such as double labeling methods or for tissues or cells that have Fc receptors (thymus, spleen).


1. How to Choose a Secondary Antibody (Sigma-Aldrich)

2. Choosing the right affinity-purified secondary antibody for your application (Jackson ImmunoResearch Laboratories)

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