Indirect detection of antigens using a secondary antibody offers several benefits over direct detection using a primary antibody:

• Using a secondary antibody enhances sensitivity and signal amplification. This is due to multiple secondary antibodies binding to a single antigen-bound primary antibody, bringing additional reporter molecules to the antibody-antigen complex.
• Primary antibodies are usually available unconjugated or conjugated to a limited range of reporter molecules. Using a secondary antibody extends access to a larger number of probes, increasing flexibility in assay design, including multiple labeling protocols. This makes secondary antibodies a much more versatile reagent as compared to individually labeled primary antibodies.
• A given secondary antibody can be used with any primary antibody of the same type and host species.
• In some cases, the same secondary antibody can be used across applications such as immunofluorescence and fluorescent western blot and immunofluorescence to validate target antigen detection.
• Using a secondary antibody prevents interference with a primary antibody’s paratope or antigen-binding site. In some cases, a primary antibody’s paratope may be compromised by conjugation to a reporter molecule, hampering its ability to bind to the protein of interest. Using a conjugated polyclonal secondary antibody to generate the signal can help preserve the primary antibody’s binding activity.
• A secondary antibody also offers the ability to perform multi-labeling or multiplexing in various assays.