Boster Bio Secondary Antibody

What is a Secondary Antibody?

Immunolabeling is critical for many areas of basic or clinical research to detect specific cell or tissue components (antigens) in a sample. This approach can be performed with direct and indirect methods by generating antigen-specific antibodies and fusing tags to antibodies.

For direct immunolabeling, the primary antibody is conjugated to a tag and directly binds to the antigen of interest. The indirect approach involves a conjugated secondary antibody that binds indirectly to the target antigen by binding to the antigen-specific primary antibody.

Secondary antibodies are generated and harvested by immunizing a host animal with an antibody from another species. For example, if the primary antibody is a rabbit polyclonal antibody, an anti-rabbit secondary antibody is raised in a different host species such as goat. The produced secondary antibody's specificity is dependent on the immunizing antibody's characteristics, such as species, subclass, fragment, etc.

Primary Antibody vs Secondary Antibody

For a successful experiment, it is important to have a good understanding of the primary and secondary antibodies. Choosing a suitable approach and/or antibodies will better serve the purpose of the experiment while saving money, time, and precious sample. Although monoclonal or polyclonal primary and secondary antibodies can be used for experiments, there are some differences between the primary and secondary antibodies that should be considered.

Primary antibodies bind directly to the antigen by recognizing a specific area/domain (epitope) of the antigen, whereas secondary antibodies bind to the primary antibody—not directly to the antigen. While primary antibodies are necessary for every immunoassay, secondary antibodies are not always required—depending on the experimental method (direct/indirect).

As previously mentioned, secondary antibodies must be made in a species different from those of the primary antibody or the specimen to minimize non-specific binding that leads to false positives and high background noise. Secondary antibodies may also require an extra purification process (pre-adsorption) with a column matrix during the immunoassay to remove non-specific antibodies and increase specificity.

Advantages of Using a Secondary Antibody

With its single labeling step, the direct method offers a shorter assay time with a simpler workflow. Since it minimizes species cross-reactivity and non-specific binding, the direct method is best used for specific targeting if multiple antibodies are raised in the same species. However, this method demands an abundant supply of expensive conjugated antibodies—with few color selections and limited range of reporter molecules available—and is much less practical than the indirect method.

Although the indirect method requires additional steps, time, and added complexity, it still offers several advantages over the direct method. More than one secondary antibody can specifically bind to different parts of the same primary antibody—increasing the versatility, antigen signal detection, and amplification. The indirect method also contributes to the detection, sorting, and purification of target antigens—providing higher degrees of specificity and sensitivity. Commercially available conjugated secondary antibodies are relatively inexpensive and available in a wider spectrum of colors compared to conjugated primary antibodies—with increased access to several different probes.

By using primary antibodies as a ‘bridge’ to bind with the target antigen, secondary antibodies reduce the possibility of the reporter molecule compromising the binding capability to the antigen epitope. If the target antigen is expressed at a low concentration, using secondary antibodies will allow for multiplexing or multi-labeling across applications (e.g. immunofluorescence, western blot) to validate target antigen detection.