Introduction to Secondary Antibodies

Full list of Secondary Antibody

Secondary antibodies are used for the indirect detection of target antigens to which a specific primary antibody is first bound. While requiring more steps than using a detectable primary antibody, indirect detection of the target antigen has the advantage of increased sensitivity due to the signal amplification from multiple secondary antibodies binding to a single primary antibody. Secondary antibodies aid in the detection, sorting or purification of target antigens by binding to the primary antibody, which directly binds to the target antigen.

Primary antibody's Fab domain binds to an antigen and exposes its Fc domain to secondary antibody. Then, secondary antibody's Fab domain binds to the primary antibody's Fc domain. Since antibody's Fc domain is constant within the same animal class, only one type of secondary antibody is required to bind to many types of primary antibodies. This reduces the cost by labeling only one type of secondary antibody, rather than labeling various types of primary antibodies.

The secondary antibody must have specificity for the antibody species and isotype of the primary antibody being used and generally has a detectable tag or other label facilitating detection or purification. Because the vast majority of primary antibodies are produced in just a few host animal species, with nearly all of the antibodies being of the IgG class, it is easy and economical for manufacturers to produce and supply ready-to-use secondary antibodies that are applicable for most methods and detection systems.

Secondary antibodies normally are polyclonal antibodies that are generated by immunizing a host animal with an antibody from a different species. For example, anti-rabbit antibodies are raised by injecting rabbit antibody into another species other than a mouse such as goat, donkey, sheep and chicken. The most common types of secondary antibodies are those generated against a pooled population of immunoglobulins from a target species. If the target antigens are total IgG, the generated secondary antibodies can bind to all classes, heavy and light chains (H&L) and fragments of target IgG, as well as other molecules sharing the same conserved domains (e.g., IgM share the same kappa light chains as IgG). In contrast, immunizing a goat with only mouse IgG1 antibodies will only generate antibodies specific for mouse IgG1 antibodies and molecules sharing the same conserved domains. Due to the high degree of conservation in the structure of many immunoglobulin domains, class-specific secondary antibodies must be affinity purified and cross-adsorbed to achieve minimal cross-reaction with other immunoglobulins. Using the example described above, immobilized mouse IgG1 antibodies would be used to affinity purify all goat antibodies that bind to mouse IgG1. These anti-mouse IgG1 antibodies would then be further purified by passage through a chromatography column(s) containing mouse IgG2a, IgG2b, IgG3, IgM, etc., to remove any antibodies that cross-react with non-IgG1 isotypes.

Additionally, secondary antibodies can be further purified by passage through columns containing the immobilized serum proteins from species other than those used to immunize the host. This method of cross-absorption (referred to as "Cross-Adsorbed") is an additional purification step recommended for applications where primary antibodies from multiple species will be used and when immunoglobulins or other serum proteins may be present in the samples being probed. For Western blot of the immunoprecipitation(IP) sample, secondary antibodies need to pass through columns containing denatured primary antibodies to minimize primary antibody (heavy and light chain) bands

According different applications, secondary antibodies are offered in three different forms: whole IgG, F(ab') 2 fragments, and Fab fragments. Moreover, in particular methods, typical secondary antibodies are either too specific (e.g., recognize only one host species of primary antibody) or too general (e.g., recognize whole IgG and any fragments thereof). Secondary antibodies are also designed to recognize whole primary antibody or particular sequences.

Anti- whole IgG (H+L)
These antibodies react with both the heavy and light chains of the IgG molecule, i.e. with both the Fc and F(ab')2 / Fab portions of IgG. Anti-IgG (H+L) antibodies also react with other immunoglobulin classes (IgM, IgA, IgD, IgE) and subclasses since they all share the same light chains (either kappa or lambda). Anti-IgG (H+L) antibodies have broader epitope recognition than anti-fragment specific antibodies. They are suggested for all general immunodetection procedures.

Anti-IgG, Fc/Fcγ fragment specific
These antibodies react with the Fc portion of the IgG heavy chain. They have been tested by ELISA and/or absorbed against Fab fragments. In some cases, they are additionally tested and/or adsorbed to minimize cross-reactivity to IgM and/ or IgA. In such cases (anti-human, anti-mouse, and anti-rat), they are labeled "Anti-IgG, Fcγ".

Caution: Anti-IgG, Fcγ fragment specific antibodies may not react equally with all monoclonal IgGs. For an anti-mouse IgG, Fcγ fragment specific antibody with balanced reactivity to four subclasses of IgG, select goat anti-mouse IgG (subclasses 1+2a+2b+3), Fcγ fragment specific (min X Hu, Bov, Rb Sr Prot).

Anti-Mouse IgG, Fcγ Subclass specific
These antibodies react with the Fc portion of the heavy chain of individual subclasses of mouse IgG. They have been tested by ELISA and/or adsorbed to minimize cross-reactivity to other subclasses, Fab fragments, IgA, IgM, and a few other species of IgG. Anti-Mouse IgG, Fcγ Subclass specific antibodies are intended for distinguishing between different subclasses of mouse IgG primary antibodies in multiple labeling experiments.

Anti-IgG, F(ab')2 fragment specific
These antibodies react with the F(ab')2 / Fab portion of IgG. They have been tested by ELISA and/or adsorbed against Fc fragments. They are not specific for IgG since they react with light chains, and therefore also react with other immunoglobulin classes (IgA, IgM, IgD, and IgE) and subclasses sharing the same light chains.

In most cases, these limitations can be overcome by carefully designing the experimental system and choosing the appropriate secondary probe. The following information can help you choose a secondary antibody for particular applications:

1. Determine the host species of the primary antibody developed
Secondary antibodies need to directly against the species of the primary antibody. Therefore, you will need a secondary antibody that is raised in a species different than the host species of the primary antibody. For example if your primary antibody is raised in a mouse, you will need an anti-mouse secondary antibody raised in goat, rabbit, etc. Select an appropriate host species for the secondary antibody (goat anti-mouse IgG, donkey anti-rabbit IgG). Some host species may not be compatible with other species in multiple-labeling experiments. In general, all secondary antibodies should come from the same host species for multiple labeling.

2. Consider cross-reactivity or specificity issues of the secondary
Antibodies from some host species may not be adsorbed against cross-reacting species of interest. Choose a host species with the required adsorptions. Highly cross-absorbed antibodies are needed for multiple-labeling applications or when using samples with endogenous antibodies

3. Specificity – binds to correct fragments, subtypes, classes or chains of the primary antibody
This question is primarily important when working with monoclonal antibodies. Polyclonal antibodies, however, are typically IgG class immunoglobulins. For this reason, the secondary antibodies will mainly be an anti-IgG antibody. In some cases, if the primary monoclonal antibody is mouse IgM, one would want a secondary antibody that reacts with mouse IgM (anti-Mouse IgM).

If the primary monoclonal is one of the mouse IgG subclasses, almost any anti-mouse IgG secondary antibody should bind to it. If the subclass of the primary antibody is not known, then anti-Mouse IgG F(ab) secondary antibodies may be used since they recognize most mouse immunoglobulin subclasses.

4. Detection or purification method
Label – appropriately conjugated to the correct enzyme, tag or fluorophore for the chosen detection method. The label is very application dependent. For immunoblotting and ELISA, enzyme-labeled secondary antibodies are the most popular. Peroxidase is economical and a more stable enzyme, in general, than alkaline phosphatase. It has also become very popular for use in chemiluminescent detection systems. Alkaline phosphatase, on the other hand, is considered more sensitive than peroxidase particularly when colorimetric detection is used. For cell or tissue staining, alkaline phosphatase, peroxidase or secondary antibodies conjugated to a fluorochrome may be used. Associated applications are immunocytochemistry/immunofluorescence, flow cytometry, or immunohistochemistry.

To generate increased amplification, a two-step biotin/avidin system may be used. Biotin binds with very high affinity to avidin resulting in an essentially irreversible interaction. A biotinylated secondary antibody is applied first, then avidin, ExtrAvidin. or streptavidin conjugated to an enzyme or fluorochrome binds to multiple sites on the biotinylated secondary antibody, thus amplifying the signal and resulting in greater sensitivity than that achieved with an antibody-enzyme or antibody-fluorochrome conjugate alone.

Ability to bind to Protein A, Protein G or Protein L – make sure the secondary antibody chosen has sufficient affinity for the molecules used upstream or downstream (i.e., Protein A-coated microplates.). Most people prefer affinity isolated secondary antibodies because they provide the lowest amount of non-specific binding. However, in certain cases, IgG fractions may be considered, i.e. when the antigen of interest is rare or present in low abundance. In these situations high affinity antibodies are required. Using pre-adsorbed antibodies will reduce non-specific background when working with tissues and cells. The process involves passing the secondary antibody over immobilized serum proteins from potentially cross reactive species. Therefore, if you are working with human tissues, choose a secondary antibody that has been adsorbed with human serum or human IgG.

5. Consider requirements of the supplied secondary
Supplied state – sterile liquid or lyophilized, suspended in PBS or Tris buffer, contains carrier proteins such as gelatin or albumin or the addition of stabilizers such as sucrose or microbial inhibitors

Goat Secondary Antibody

Goat Anti-Human IgG-Fc Antibody Goat Anti-Rat IgG Antibody
Goat Anti-Human IgG Antibody Goat Anti-Mouse IgG-Fc Antibody
Goat Anti-Rabbit IgG-Fc Antibody Goat Anti-Mouse IgG Antibody
Goat Anti-Rabbit IgG Antibody  

Rabbit Secondary Antibody

Rabbit Anti-Mouse IgM Antibody Rabbit Anti-Human IgG Fc Antibody
Rabbit Anti-Mouse IgG Antibody  

Mouse Secondary Antibody

Mouse Anti-Human IgG Antibody Mouse Anti-Human IgG Fc Antibody

HRP Labeled Secondary Antibody

Goat Anti-Human IgG-Fc Antibody Goat Anti-Mouse IgG Antibody
Goat Anti-Human IgG Antibody Rabbit Anti-Mouse IgM Antibody
Goat Anti-Rabbit IgG-Fc Antibody Rabbit Anti-Mouse IgG Antibody
Goat Anti-Rabbit IgG Antibody Mouse Anti-Human IgG Antibody
Goat Anti-Rat IgG Antibody Mouse Anti-Human IgG Fc Antibody
Goat Anti-Mouse IgG-Fc Antibody Rabbit Anti-Human IgG Fc Antibody

Biotin Labeled Secondary Antibody

Goat Anti-Human IgG-Fc Antibody Goat Anti-Mouse IgG Antibody
Goat Anti-Human IgG Antibody Rabbit Anti-Mouse IgM Antibody
Goat Anti-Rabbit IgG-Fc Antibody Rabbit Anti-Mouse IgG Antibody
Goat Anti-Rabbit IgG Antibody Mouse Anti-Human IgG Antibody
Goat Anti-Rat IgG Antibody Mouse Anti-Human IgG Fc Antibody
Goat Anti-Mouse IgG-Fc Antibody Rabbit Anti-Human IgG Fc Antibody

ALP Labeled Secondary Antibody

Goat Anti-Human IgG-Fc Antibody Goat Anti-Mouse IgG Antibody
Goat Anti-Human IgG Antibody Rabbit Anti-Mouse IgM Antibody
Goat Anti-Rabbit IgG-Fc Antibody Rabbit Anti-Mouse IgG Antibody
Goat Anti-Rabbit IgG Antibody Mouse Anti-Human IgG Antibody
Goat Anti-Rat IgG Antibody Mouse Anti-Human IgG Fc Antibody
Goat Anti-Mouse IgG-Fc Antibody Rabbit Anti-Human IgG Fc Antibody

FITC Labeled Secondary Antibody

Goat Anti-Human IgG-Fc Antibody Goat Anti-Mouse IgG Antibody
Goat Anti-Human IgG Antibody Rabbit Anti-Mouse IgM Antibody
Goat Anti-Rabbit IgG-Fc Antibody Rabbit Anti-Mouse IgG Antibody
Goat Anti-Rabbit IgG Antibody Mouse Anti-Human IgG Antibody
Goat Anti-Rat IgG Antibody Mouse Anti-Human IgG Fc Antibody
Goat Anti-Mouse IgG-Fc Antibody Rabbit Anti-Human IgG Fc Antibody

PE Labeled Secondary Antibody

Goat Anti-Human IgG-Fc Antibody Goat Anti-Mouse IgG Antibody
Goat Anti-Human IgG Antibody Rabbit Anti-Mouse IgM Antibody
Goat Anti-Rabbit IgG-Fc Antibody Rabbit Anti-Mouse IgG Antibody
Goat Anti-Rabbit IgG Antibody Mouse Anti-Human IgG Antibody
Goat Anti-Rat IgG Antibody Mouse Anti-Human IgG Fc Antibody
Goat Anti-Mouse IgG-Fc Antibody Rabbit Anti-Human IgG Fc Antibody

APC Labeled Secondary Antibody

Goat Anti-Human IgG-Fc Antibody Goat Anti-Mouse IgG Antibody
Goat Anti-Human IgG Antibody Rabbit Anti-Mouse IgM Antibody
Goat Anti-Rabbit IgG-Fc Antibody Rabbit Anti-Mouse IgG Antibody
Goat Anti-Rabbit IgG Antibody Mouse Anti-Human IgG Antibody
Goat Anti-Rat IgG Antibody Mouse Anti-Human IgG Fc Antibody
Goat Anti-Mouse IgG-Fc Antibody Rabbit Anti-Human IgG Fc Antibody

Antibody

Flow Cytometry (FCM) / FACS Antibody
- Flow Cytometry (FCM) / FACS Antibody Features
- Flow Cytometry (FCM) / FACS Products Center
- Flow Cytometry (FCM) / FACS Technology Center