Secondary antibodies bind to the primary antibody to assist in detection, sorting and purification of target antigens.
To enable detection, the secondary antibody must have specificity for the antibody species and isotype of the primary antibody being used and generally is conjugated.
Secondary antibodies are used throughout various types of assays, including ELISA or Western Blot, Immunohistochemistry, Flow Cytometry. The secondary antibody type is selected according to the class of the primary antibody (e.g., IgG or IgM), the source host, and the kind of label which is preferred. Most primary antibodies are of the IgG class and are produced in a common set of host species that includes rabbit, mouse, goat or chicken. Therefore, anti-mouse IgG, anti-rabbit IgG, anti-goat IgG or anti-chicken polyclonal antibodies are often used.
Choosing a secondary antibody is straightforward: select a secondary antibody that recognizes the host species used to produce the primary antibody of interest, i.e. choose an anti-mouse secondary antibody to detect a mouse monoclonal primary antibody. However, identifying the optimal secondary antibody requires knowledge of the detection assay. For example: Western blot and ELISA can be performed using colorimetric, chemiluminescent, and fluorescence reporter systems, while immunofluorescence and flow cytometry are generally limited to fluorescent reporter labels. In all of these instances, a conjugated secondary antibodyis required.