Secondary Antibody: How to Choose

Secondary Antibody: How to Choose

With so many antibodies on the market, this article “Secondary Antibody: How to Choose” will help a scientist better understand secondary antibodies and give the tools to  choose the best antibody. The main function of the secondary antibody is to bind to the primary antibody. For example, if the primary antibody is a monoclonal antibody of mouse origin, the secondary antibody must bind to mouse antibodies. In the example below, the secondary antibody must be an “anti-Mouse IgG”. See Figure 1. If the primary is prepared from rabbit, an “anti-Rabbit IgG” secondary antibody must be used.

Indirect ELISA with Goat anti-Mouse Secondary

Figure 1







Monoclonal primary antibodies that are derived from mice (most typical) are of a single subclass: IgG1, IgG2a, IgG2b, etc. Sometimes it may be advantageous to use a secondary antibody that binds only to that subclass. For example, if the primary antibody is an IgG1 subclass, you could choose a secondary that binds specifically to IgG1. (i.e.Goat anti-Mouse IgG1)This is particularly suited when more than one primary antibody is being used in double staining applications.

If the primary antibody is an IgM or IgA class (aka isotype), you will choose a secondary antibody that recognizes IgM or IgA. (i.e. Goat anti-Mouse IgM-HRP)

IR TIP- Rabbits only have a single subclass. No subclass specific antibody is needed.  

Another factor to consider that is equally important is the “cross-absorption” level of the secondary antibody. While secondary antibodies react with primary antibodies, they can also react with the host sample that you are detecting in your assay. See Figure 2.  This can lead to false positives, splotchy images, and overall poor results. To minimize this problem, the scientist should choose a secondary antibody that has been cross-absorbed (aka cross-adsorbed) against the host sample. For example, if the scientist is testing rat spleen cells, the secondary antibody should be cross absorbed against rat serum proteins. IR-Tip- ImmunoReagents denotes their cross-absorbed secondary antibodies as: (min-x Rat). This means “minimum cross-reactivity to rat”.


Figure 2 Secondary Antibody Binding the Primary Antibody and Host Cell








The final factor in choosing a secondary antibody is if the assay requires a conjugated (aka labeled) antibody. Most immunoassays (ELISA’s, Western Blots, Immunohistochemistry, Flow Cytometry) require a conjugated antibody. For Western Blot and ELISA assays, the most commonly used conjugate is Horseradish Peroxidase (HRP) and Alkaline Phosphatase (Alk-Phos). Immunoassays that use florescence will require a secondary that is conjugated with a fluorescent dye. The most popular fluorescent dyes are FITC, TRITC, along with DyLight® dyes. For more information on choosing a Conjugated Secondary Antibody click here.

ImmunoReagents, Inc. is a leading global manufacturer of quality antibodies and reagents used in pharmaceutical research, life science research and in vitro diagnostics. Product offerings include a wide range of immunochemistry reagents such as purified immunoglobulins, primary antibodies, and secondary antibodies covering a broad spectrum of immunoglobulins from various species. ImmunoReagents also provides custom manufacturing to meet specific customer requirements while adhering to cGMP guidelines and ISO quality systems requirements. The company is located in Raleigh, North Carolina

For any help is choosing the appropriate secondary antibody please contact ImmunoReagents technical support team: [email protected]  ImmunoReagents is a manufacturer of nearly 2000 secondary antibodies.