Overview of Primary Antibodies and Secondary Antibodies

Detecting a target of interest in studies of, for example, biochemistry, cell biology, and histology often involves the use of a primary antibody to specifically bind to the target. A secondary antibody is employed that binds to the primary antibody-target complex to capture the complex and to deliver a means of detecting the complex.

What is a Primary Antibody?

The primary antibody is raised against an antigen representing the target of interest. A unique antigen must be used that potentially confers no cross-reactivity of the Primary Antibody with other targets, eliminating the possibility of reactive noise or a low signal-to-noise ratio in the desired application.

The primary antibody is the one that binds directly to the target. The variable region of the primary antibody recognizes an epitope on the target. It is produced by a host organism that is of a different species than the specimen. The primary antibody usually does not contain a fluorophore or an enzyme, so the researcher cannot visualize the target without further reagents such as a secondary antibody.

What is a Secondary Antibody?

The secondary antibody binds to the primary antibody but not any target that is present in the specimen. Secondary antibodies bind to the heavy chains of primary antibodies so that they don’t interfere with the primary antibody binding to the target. This secondary antibody is made in a species that is different than both those of the primary antibody or the specimen. This minimizes non-specific binding that leads to false positives and high background noise.

Why Use Two Antibodies?

An antibody may work very well for multiple applications, such as western blotting and immunofluorescence, but different assays have experimental conditions that require different methods of visualization. Using the same primary antibody in multiple different assays often requires a secondary antibody. The primary antibody detects the antigen in the specimen, but the secondary antibody can be designed to have a fluorophore or enzyme complex attached to it for the purposes of visualization.

Dual Labeling Using Fluorescence

The use of two antibodies to visualize an antigen provides the advantage of picking what color will represent the location of the antigen. Secondary antibodies can be engineered to carry different-colored fluorophores (i.e. green, red). Thus, the antigen bound by a primary antibody can appear green in one experiment, but red in a different experiment, depending on what secondary antibodies are available. The primary-secondary antibody system for dual-labeling of distinct antigens in the same specimen produces cleaner data and allows for more flexibility regarding what secondary antibodies in your freezer will work for the question that you are asking.

Using Binding Systems That Are Stronger Than the Attraction Between Antibodies

Antibodies bind very tightly to their antigens, but the strength of this binding depends on the stability of the antibody (i.e. temperature, pH). The strongest non-covalent binding between a protein and its ligand is between biotin and avidin. The strength of the bond between these two molecules is more resistant to washing steps. The tightness also reduces the amount of non-specific binding. Using a primary-secondary antibody system allows researchers to employ the strength of the biotin-avidin bond is targeting a colorful chemical to the location of an antigen.