How to select the right secondary antibody

Secondary antibodies are used in several immunoassays to detect the presence of the primary antibodies. These antibodies are conjugated to enzymes, biomolecules, or fluorescent dyes to allow detection of the primary antibodies. Unlike primary antibodies, secondary antibodies are raised against the species and isotype of the primary antibody, and are used to detect the primary antibody by binding to it in more than one place.

To successfully choose your secondary antibody, you should consider the following factors:

Ensure a good match – The host species is the animal in which the secondary antibody was generated and should always be different from the host of the primary antibody. For example, if you use a primary antibody raised in rabbit, you will need an anti-rabbit secondary antibody raised in a different species other than rabbit, such as goat, donkey, or mouse (Figure 1).


Figure 1: Secondary antibody mechanism.

Whole antibodies or fragments? – Actually, the real question would be “is your primary antibody monoclonal or polyclonal?”  The answer to both questions is often dependent on the application. Monoclonal primary antibodies contain a single isotype of immunoglobulin. Therefore, it is critical to use a secondary antibody that specifically recognizes that isotype. Also, monoclonals are usually raised in mouse, rabbit, and rat. As such, if the primary monoclonal antibody is a mouse IgG1, you will need an anti-mouse IgG or a less specific F(ab) fragment anti-mouse IgG. Antibody fragments are best for immunohistochemistry and immunofluorescence because of their smaller size and ability to penetrate tissues. However, the smaller size may limit the choice of dyes or enzymes to be conjugated to the fragments, resulting in secondary antibodies that may be less sensitive than whole antibody options.

Polyclonal primary antibodies contain a mixture of several isotypes of immunoglobulins G. Therefore, to maximize detection of the target, it is best to use a secondary antibody that recognizes all isotypes. Polyclonals are usually raised in rabbit, goat, sheep, and donkey and in whole antibody format (Heavy + Light chains), so the secondary host species must be immunized with a pool of IgG’s from a different species, thereby allowing the purified secondary antibodies to recognize all forms. For example, if the primary polyclonal antibody is a goat IgG, you will need an anti-goat IgG (H+L). However, whole antibodies can result in high background and lower specificity because the light chain is shared by all immunoglobulins, which increases cross-reactivity (Figure2).


Figure 2: Binding mechanism of a Whole IgG secondary antibody versus a Fragmented secondary antibody in the immunoassay.

Choose the conjugate according to your application - This depends on how the secondary antibody detects the signal. For example, immunohistochemistry, ELISA, and western blot applications use colorimetric and chemiluminescent detections based on Biotin labelling to amplify the signal and increase sensitivity, and enzymatic reactions acting with horseradish peroxidase (HRP), or alkaline phosphatase (AP). For immunolabeling, fluorescence microscopy and flow cytometry experiments, however, the signal is detected using fluorescent dyes such as Alexa Fluor, which are conjugated directly to the antibodies to amplify the signal and sensitivity.

Affinity purified vs. cross-adsorbed antibodies – This step is used to increase the specificity of the antibodies and can be distinguished in affinity purification and cross-adsorption. Most secondary antibodies are purified through affinity chromatography, resulting in a high affinity antibody. Antibodies of this kind are commonly used in IHC because they give minimal non-specific binding, as well as in western blot to detect low-abundance proteins. Cross-adsorbed secondary antibodies often go through an additional purification step to filter out members that bind to off-target species of immunoglobulin. This step increases their specificity and reduces non-specific background. These antibodies are designed for particular applications such as immunohistochemistry.

In summary, secondary antibody choices depend on the application at hand and the level of sensitivity and purification required with respect to target abundance, as well as the risk of non-specific binding. If you would like to discuss the approaches introduced above, you are always welcome to contact us!

Author: Dr. Sonia Accossato

Copyright © BMA Biomedicals 2022