A secondary antibody aids in the detection, sorting or purification of target antigens by binding to the primary antibody, which directly binds to the target antigen. Since, 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. There are many secondary antibody options available for purity level, specificity and label type no matter the application.

Video summary of secondary antibodies

Watch this video on using secondary antibodies for signal amplification

Advantages of using secondary antibodies

Secondary antibodies are used for the indirect detection of a target to which a specific primary antibody is first bound. The secondary antibody must have specificity both for the antibody species as well as the isotype of the primary antibody being used. Also, a secondary antibody generally has a detectable tag or other label facilitating detection or purification.

Indirect detection of the target antigen using secondary antibodies requires more steps than direct detection using primary antibodies. However, the advantage of indirect detection is increased sensitivity due to the signal amplification from multiple secondary antibodies binding to a single primary antibody. In addition, a given secondary antibody can be used with any primary antibody of the same type and host species, making it an infinitely more versatile reagent than individually labeled primary antibodies. Secondary antibodies with specificity for the primary antibodies of common species are commercially available pre-conjugated with many of the common labels, including fluorescent and enzyme conjugate options.

Difference between direct and indirect protein detection in an assay involving detection with specific antibodies (e.g., an immunoassay). The surface on which the target protein is bound and immobilized is either a membrane (western blot) or a microplate well (ELISA).


Indirect detection of antigens using secondary antibodies offers other advantages besides diversity, flexibility, and versatility:

  • Allow for the use of the same primary antibody with different secondary antibodies depending on the application.
  • In some cases the same secondary antibody can be used across applications (i.e. fluorescent western blot and immunofluorescence) to validate target antigen detection.
  • Secondary antibodies also offer the ability to perform multiplexing or multi-labeling in experiments.

Specificity of secondary antibodies

Secondary antibodies are generated by immunizing a host animal with the antibody(s) from a different species. For example, anti-mouse secondary antibodies are raised by injecting mouse antibodies into an animal other than a mouse. Goat, donkey and rabbit are the most commonly used host species for producing secondary antibodies, but other host species may also be used.

The most common types of secondary antibodies are those generated against a pooled population of immunoglobulins from a target species. For example, immunizing a goat with purified mouse IgG will generate goat anti-mouse IgG antibodies that will bind to all classes, heavy and light chains (H+L) and fragments of mouse IgG as well as any 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.

Because of 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 immobilized serum proteins from species other than those used to immunize the host. This method of cross-adsorption (often referred to as "Highly 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.


Secondary antibody formats

Secondary antibodies are available in 3 formats:

  • Ready-to-use liquid
  • Lyophilized
  • With additives, such as glycerol

The secondary antibody format depends on how the antibody is produced and how secondary antibody providers may choose to ship their product. Ready-to-use liquid secondary antibodies are supplied as concentrates that may need some optimization in order to determine their optimal working conditions. The product data sheet often provides recommended dilutions. Ready-to-use liquid antibody concentrates need to be stored between 4 and 8°C and are stable for up to a year if stored according to the manufacturer’s instructions.  Ready-to-use liquid antibodies can be stored as aliquots at –20°C or lower to extend their shelf life. Aliquots can then be thawed to prepare a working solution immediately before use. Aliquoting prior to storage at –20°C is recommended for ready-to-use secondary antibodies as repetitive freezing and thawing leads to a loss of staining capacity.

Dry lyophilized concentrated secondary antibody stocks have to be reconstituted with diluents prior to use. While vials of dry, lyophilized antibodies are stable in that format for years at –20°C or lower but after reconstitution, they have to be treated like liquid secondary antibodies.

Glycerol is used as an additive in antibody stock solutions to extend their shelf life during storage at –80 to –20°C. Another common additive in commercially available antibodies is BSA, which is used as a stabilizer. Since compatibility of the antibody solution may not have been a key consideration when the antibody was initially purified and formulated, it is important to consider the additives present in the antibody stock solutions as different labeling methods may be impacted to different extents by the various additives.

Characteristics to consider when choosing a secondary antibody format are summarized here:

Ready-to-use liquid Lyophilized Liquid/lyophilized with additives
Liquid secondary antibodies come ready to use out of the vial. Lyophilized secondary antibodies need to be reconstituted properly in the right buffer and pH. Secondary antibody additives such as BSA, glycerol, etc. can interfere with target detection, depending on the application.
Liquid secondary antibodies are ready to aliquot for long term storage. Lyophilized secondary antibodies could potentially have performance or functional loss if antibodies are not properly reconstituted. Secondary antibodies with additives may need to be tested at various dilutions to ensure that the antibody functions properly with the additives.

The secondary antibody format one chooses to buy will therefore depend on many factors including where the antibody is purchased, the type of experiment being performed, or storage time before use. Invitrogen secondary antibodies are available in the ready-to-use liquid format for ease of use and storage.


Using secondary antibodies

Secondary antibodies are available conjugated to labels and also in unconjugated forms. The choice of the label depends upon the application and how the secondary antibody is going to be detected. Enzyme labels are visualized via chromogenic reactions. Commonly used enzymes include horseradish peroxidase (HRP) and alkaline phosphatase. HRP substrates are more stable and relatively inexpensive. Alkaline phosphatase provides longer lasting signal (24–48 hr) and is more sensitive than HRP for colorimetric detection. Avidin-Biotin systems are used when signal amplification is desired while detecting proteins expressed at low levels. Secondary antibodies conjugated to fluorescent-dyes provide brighter signals, multiplexing capabilities and ease of use.

Invitrogen secondary antibody conjugates include:

Since secondary antibodies can be conjugated to a large number of labels, the type of secondary antibody used ultimately depends on the downstream application.

The table below summarizes the types of secondary antibodies used in some common applications:

Application Types of secondary antibodies used
Western blot
  • Enzyme conjugated (HRP or AP) secondary antibodies are most commonly used
  • Fluorescently labeled secondary antibodies are also available
  • Sensitivity of fluorescence-based detection is similar to that of chemiluminescence detection
  • Fluorescence-based detection allows for the detection of multiple fluorophores at the same time

ELISA

  • Most commercial kits use streptavidin-HRP (biotinylated secondary) antibodies
  • Give stronger signal than conventional enzyme-labeled secondary antibodies

Immunohistochemistry

  • Secondary antibodies conjugated to enzymes
  • Biotinylated secondary antibodies

Immunocytochemistry

  • Secondary antibodies conjugated to enzymes
  • Biotinylated secondary antibodies
  • Fluorescently labeled secondary antibodies
  • Poly-HRP–mediated tyramide signal amplification

Immunofluorescence

  • Fluorescently labeled secondary antibodies
  • Poly-HRP–mediated tyramide signal amplification

Flow cytometry

  • Fluorescently labeled secondary antibodies
  • Secondary antibodies conjugated to enzymes
  • Biotinylated secondary antibodies