Secondary antibodies (secondaries) are generated by immunizing a host animal with the antibody(s) from a different species. Secondary antibodies†are available with specificity for whole Ig molecules or antibody fragments such as the Fc or Fab regions. These antibodies are typically labeled with probes that make them useful for detection, purification or sorting applications. Secondary antibodies may be polyclonal or monoclonal. Polyclonals lack the specificity of monoclonals but frequently have higher sensitivities because polyclonals are a mixture of antibodies that may include very high affinity antibodies. Cloning may not effectively select the highest affinity antibody from a polyclonal host.
Anti-mouse 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 raising secondary antibodies, but others may be available from individual suppliers. The most common types of secondary antibodies are those generated against a pooled population of immunoglobulins from a target species. 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-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 antibodies are used in immunodetection and immunoaffinity purification applications. The specific utility of a secondary antibody depends upon its conjugated probe(s). Probes are molecules that support various detection technologies. The most common detection systems for conjugated secondary antibody are colorimetric†and fluorescent. Immunodetection applications include ELISA, Western blotting, Immunohistology, Immunoblotting, Immunostaining and cell based assays, such as cell-based immunochemical assays and high throughput cell-based screening assays (HTS). Secondary antibodies are also frequently utilized in flow cytometry†for cell sorting. Colorometric assays are typically based on the use of alkaline phosphatase (AP) or horseradish peroxidase (HRP) or derivatives of these, APAAP or PAP. The biotin avidin (streptavidin) conjugate binding system is often used to amplify the colorometric signal for AP or HRP-conjugated secondary. The most common fluorescent assays utilize Fluorescein (FITC) or Rhodamine or it derivative, TRITC. Newer fluorescent dyes such as Cyanine (Cy3) and Phycoerythrin (R-PE), which have unique advantages, are also used. A secondary antibody can also be conjugated with agarose or colloidal gold for special applications. There are a number of reasons for using a labeled secondary antibody†instead of a labeled primary antibody. Understanding the structure and specificity requirements of the intended assay system is essential to choosing an appropriate secondary antibody and obtaining the best possible results. When samples contain few sources of cross-reactivity, choosing a secondary antibody that recognizes a whole immunoglobulin molecule generally will provide the most signal. For samples containing endogenous immunoglobulinís, using a secondary antibody that is fragment-specific to the primary antibody will provide the most specific signal. Still other applications, such as IHC, produce the best result when the specific secondary antibody is itself a fragment.
Secondary antibodies provide several advantages over direct detection with labeled primaries. Most importantly, primaries are typically expensive to buy or produce, and researchers may not wish to risk poor recovery or complete inactivation by subjecting the antibody to a labeling procedure to make it detectable. †While requiring more steps than using a detectable primary antibody, indirect detection of the target antigen with a secondary†has the advantage of 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 a more versatile reagent than individual labeled primaries. Secondary antibodies with specificity for the primary antibodies of common species are commercially available pre-conjugated with many of the common labels, making these detection reagents commodities.
∑† View a list of anti-mouse Secondary Antibodies
∑† View a list of anti-rabbit Secondary Antibodies
∑† View a list of anti-rat Secondary Antibodies
∑† View a list of anti-hamster Secondary Antibodies
∑† View a list of anti-goat Secondary Antibodies