Secondary Antibodies & Reagents

When selecting secondary antibodies, it is important to consider the host species of your primary antibody and the type of readout you wish to use. For example, if your primary antibody is a rabbit polyclonal and your intention is to perform a western blot with chemiluminescent detection, you will likely want to choose an anti-rabbit IgG secondary antibody that detects both heavy and light chains, is labeled with horseradish peroxidase (HRP), and is validated for the western blot application. Likewise, if you are planning to combine several mouse monoclonal primary antibodies of different subclasses in a multiplexed ICC experiment, you will want to look for subclass-specific secondary antibodies that are labeled with spectrally distinct fluorescent dyes and shown to work for ICC.

Secondary antibodies that have been affinity purified and/or cross-adsorbed have a reduced risk of non-specific binding that can cause unwanted background signal. During affinity purification, the primary antibody of interest is immobilized and used to isolate target-specific secondary antibodies from a heterogeneous antibody mixture. For example, immobilized mouse IgG2a antibodies are used to affinity purify goat anti-mouse IgG2a. Cross-adsorption works on a similar basis and serves to remove any antibodies demonstrating undesirable off-target binding, either to antibodies from another species or to antibodies of a different class or subclass.

Affinity purified and/or cross-adsorbed secondary antibodies are especially useful for applications such as sandwich ELISA, where secondary antibody binding to the immobilized capture antibody must be avoided, and IHC, where it is critical that the secondary antibody does not bind non-specifically to the sample material.

Full-length antibody molecules have two antigen-binding fragments (Fabs) and a crystallizable fragment (Fc), of which the latter functions to interact with Fc receptors on the surface of immune cells during an immune response. Secondary antibody fragments such as Fab and F(ab')2 lack the Fc region, preventing them from binding non-specifically to Fc receptors in applications such as flow cytometry, IHC, and ICC. Another advantage of using secondary antibody fragments for research is that their small size allows for better penetration into cells and tissues, which can improve assay sensitivity.

Because biotinylated primary or secondary antibodies do not produce a measurable signal, labeled secondary reagents are required for detection. These usually consist of avidin or streptavidin that has been conjugated to an enzyme or fluorescent dye. Because each biotinylated antibody features multiple biotin molecules, and each avidin/streptavidin molecule can bind four biotins, an advantage of this approach is that it provides signal amplification. Neutralite avidin is a modified version of avidin which is both neutral and deglycosylated to reduce non-specific binding.