Secondary and primary antibodies share a common structure. The antibodies are all y-type, with three parts roughly the same size. They are connected by two identical light chains and two identical heavy chains forming - disulfide bonds. Secondary antibody is bound to the primary antibody attached to the antigen. The labeled secondary antibody binds their V region to the stem or C region of the primary antibody. Using the labeled secondary antibodies, it can indirectly aid in the detection and purification of proteins. The application of secondary antibodies improves the sensitivity in labeling and detection.

They were used for the colorimetric, chemiluminescence, and fluorescence detection of primary antibodies for a range of applications, such as flow cytometry, immunoblotting, and cellular imaging.