Secondary Antibody Selection

How to choose the best secondary antibody for your application

In many biological assays, the target antigen is detected indirectly by secondary antibodies which bind to primary antibodies in multiple places to allow signal detection and amplification. The choice of appropriate secondary antibody is determined by the specifications (i.e. species, subclass, and fragment type) of the primary antibody and the application. Secondary antibodies are available conjugated to various fluorescent dyes (e.g. Proteintech's CoraLite dyes), and enzymes (e.g. horseradish peroxidase). 

In this article, we set out the key points to consider when choosing an appropriate secondary antibody for your application. 



1. What is the host species of your primary antibody?

The secondary antibody must be raised against the host species of the primary antibody. For example, if the primary antibody was generated in mouse, the secondary needs to be anti-mouse and raised in a host species other than mouse (e.g. goat anti-mouse secondary antibody). 


Figure 1. Most common host species of primary antibody.


2. Is your primary antibody polyclonal or monoclonal?

Polyclonal antibody

A polyclonal antibody contains a mixture of several isotypes of immunoglobulin G (e.g. IgG1, IgG2a). Therefore, to maximize detection of the target, it is best to use a secondary antibody that recognizes all isotypes. These are any secondary antibodies which are labeled “IgG H+L” (H and L represent heavy and light chains of IgG, respectively). This designation means the secondary host species was immunized with a pool of IgG’s from another species allowing the purified secondary antibody to recognize all forms.


Monoclonal antibody

A monoclonal antibody contains a single isotype of immunoglobulin (e.g. mouse IgG2a), which should be indicated on the primary antibody product datasheet. When selecting a secondary antibody to detect a monoclonal primary antibody, it is possible to use a class specific (e.g. anti-mouse IgG), or a subclass specific secondary antibody (e.g. anti-mouse IgG2a).



Figure 2. Categories of antibodies: A) polyclonal antibodies are binding to the same antigen, but different epitopes and B) monoclonal antibodies are binding to the same epitope on a target antigen. Please note: If the primary antibody is a fragment (not the whole antibody), the secondary antibody should also be fragment-specific to reduce noise signal (e.g., F(ab’)₂ fragment secondary antibodies penetrate tissue easier in IHC).


3. What is your method of signal detection?

Secondary antibodies can be conjugated to a wide range of chemicals, including enzymes (horseradish peroxidase (HRP) or alkaline phosphatase (AP)) and fluorophores (e.g. fluorescein (FITC) or Cyanine (Cy3)).


Secondary antibody conjugates
Western blot
  • Enzyme conjugates, e.g. HRP for chemiluminescent detection and AP for colorimetric detection
  • Fluorophores for fluorescent detection
  • Enzyme conjugates, e.g. Polymer HRP
  • Biotinylated for signal amplification
  • Fluorophores
Flow cytometry
  • Fluorophores and fluorescent dyes
  • Enzyme conjugates
  • Biotinylated, with fluorescent-labeled streptavidin
  • Biotinylated with streptavidin-HRP
  • Enzyme conjugates, e.g. HRP


4. Other considerations


When selecting a secondary antibody, it is important to consider how the antibody has been purified. A common purification technique for secondary antibodies is High-Affinity Purification, resulting in reduced non-specific binding. In addition, secondary antibodies can go through an additional purification step to reduce potential cross-reactions with other species called cross-adsorption. In this process, the secondary antibody solution is passed over different columns containing sera proteins of different species to filter out the non-specific secondary antibody (e.g., for multi-staining).


Western Blotting after Immunoprecipitation

When using Western blot to detect a protein in immunoprecipitated samples, extra care should be given to the choice of antibodies. Immunoprecipitated samples contain the pull-down antibody’s heavy and light chains. These chains show up at 50 kDa and 25 kDa. If the secondary antibody recognizes the pull-down antibody and the Western blot’s primary antibody these bands will show up at a high intensity and may overshadow the target protein signal. It is best to use a different species for the Western Blot’s primary antibody than the pull-down antibody. If this is not possible it is recommended to use a secondary that recognizes the specific chain furthest away from your target protein or to use a secondary antibody that prefers the native over denatured forms.