Antibodies are invaluable to scientific research, diagnostics, and therapeutics. These simple yet powerful scientific tools find their way into a diverse range of fields and applications. Secondary antibodies provide a mechanism for analyte detection without inferring with its recognition and provide inherent signal amplification. This article briefly describes secondary antibodies.

What is a secondary antibody?

A secondary antibody is an antibody, much like any other; its nomenclature stems from its place in the application or experimental setup. The secondary antibody is used to detect the antibody specific for the protein of interest (POI) – the primary antibody. Predominantly the secondary antibody is conjugated to the detection reagent such as fluorophore or reporter enzyme but could be an unconjugated “bridging” antibody used in an ELISA. Of course, a secondary antibody could be an antibody that isn’t specific to the primary antibody but could be a tertiary antibody in the experimental setup.

Direct and indirect detection

Fig 1: A: Direct IHC, B: Indirect IHC, C: Signal enhancement with biotinylated secondary and labeled streptavidin

A protein of interest (antigen) is specifically recognized by a primary antibody, which may be directly conjugated (direct detection, Fig 1.A); or the primary may itself be detected by a conjugated secondary antibody (indirect detection, Fig 1.B), allowing visualization of the protein/analyte.

Why use secondary antibodies?

The indirect method using a secondary antibody offers many advantages over the direct method.

• Inherent signal enhancement – multiple secondary antibodies bind to one antigen-bound primary antibody, bringing additional reporter molecules to the antigen-antibody complex enhancing signal (Fig 1: B). The addition of a biotinylated secondary antibody followed by conjugated streptavidin can be used to increase signal further (Fig 1:C).

• Expanded conjugate availability – the dye availability of conjugated primaries may be limited, by using a secondary antibody many more conjugates can be made available increasing the options for multiple labeling experiments.

• Prevents interference with paratope – By using a conjugated secondary antibody, the antigen binding site (paratope) on the primary antibody is preserved.

Learn more about choosing your secondary antibody here