AAT Bioquest

How do I choose secondary antibodies?

Posted September 22, 2021


Choosing the correct secondary antibody is crucial for the successful detection of the target antigen. These are some of the factors to consider when choosing a secondary antibody:

  • Host & Target Species: The first step is to determine which host species was used to generate the primary antibody. The secondary antibody that you select must be specific for detection of the primary antibody species. The species used to generate the secondary antibody cannot be the same as the host species of the primary antibody.
  • Purity Level & Pre-Adsorption Of The Secondary Antibody: Affinity purified secondary antibodies are the preferred option. This is because they reduce background and increase specificity, and sensitivity. They also improve lot-to-lot consistency leading to more reproducible immunoassays. It is also preferable to use a pre-adsorbed secondary antibody. Pre-adsorbed secondary antibodies improve specificity and sensitivity by eliminating reactivity from immunoglobulins of undesired cell and tissue samples, antibody fragments, or species. 
  • Type Of Detection Assay: Different applications require differe:nt types of secondary antibodies. An enzyme conjugated secondary antibody is the preferred choice for applications such as Western blot and ELISA. A secondary antibody conjugated to a fluorochrome is the preferred choice for immunoassays such as flow cytometry or immunofluorescence microscopy.
  • Class/Subclass Of The Primary Antibody: Primary monoclonal antibodies are usually IgG isotype with a specific subclass. The secondary antibody that you select must be directed against that specific subclass (anti-IgG). If the primary polyclonal antibody has more than one subclass, the anti-IgG must be able to recognize both the heavy and light IgG chains.
  • Whole Or Fragmented Antibodies: Whole secondary antibodies containing both heavy and light (H &L) chains of Ig are more commonly used because they give a higher signal. This is because they bind more strongly to variable regions. However, whole antibodies also tend to increase cross-reactivity and reduce specificity. Because of this, for some applications such as immunofluorescence, flow cytometry, and immunohistochemistry (IHC), it may be preferable to use a fragment conjugated secondary antibody to eliminate non-specific binding.
Additional resources

Benefits and pitfalls of secondary antibodies: why choosing the right secondary is of primary importance

Power Styramide™ Signal Amplification (PSA™)

iFluor 350™ PSA™ Imaging Kit with Goat Anti-Mouse IgG