A Guide to Secondary Antibodies

In laboratory applications, secondary antibodies are reagents intended to bind primary antibodies and produce or amplify a signal for detection. They are crucial in the indirect method of immunodetection utilized in techniques such as ELISA, Western blotting, flow cytometry, immunohistochemistry, and fluorescence microscopy. Secondary antibodies are meant to be paired with primary antibodies, which function by specifically binding proteins and antigens of interest. A proper pairing of a primary and secondary antibody can amplify the signal of even low abundance target antigens. The success and quality of immunodetection experiments will heavily rely not only primary antibodies, but also on the chosen secondary antibodies.

Advantages of secondary antibodies

Signal amplification is one key advantage of secondary antibodies. Multiple secondary antibodies can bind to a single primary antibody, increasing the overall signal and making it easier to detect the target antigen, especially those that are in low abundance.

Another advantage is versatility. Because secondary antibodies target immunoglobulins, they can be used with any primary antibody of the appropriate species and isotype. Secondary antibodies are also independently labeled, meaning the detection method can be changed, while keeping the primary antibody the same, or vice versa. This is also helpful when multiple primary antibodies are being used for different targets (i.e. multiplexing), as having different labeled secondary antibodies provides more options when designing panels.

General properties of secondary antibodies

While secondary and primary antibodies are generally structurally similar, the defining characteristic that defines an antibody as a “secondary” is its affinity and specificity to a primary antibody. Polyclonal secondary antibodies are produced by immunizing a host animal with a whole or fragmented immunoglobulin from an animal of a different species. Monoclonal secondary antibodies use the same principle but utilize a B cell hybridoma fusion to produce a clonal population of species-specific antibodies.

Another key feature of secondary antibodies is the attachment of a label. Detection labels are often conjugated to secondary antibodies to generate a signal for detection. Enzyme labels, fluorophores, and biotin are available, which offer users flexibility for different applications. Common labels are highlighted below:

Horseradish peroxidase (HRP): HRP is a 44-kDa enzyme that reacts with colorless, chromogenic substrates (eg. TMB, DAB, ABTS) to produce a detectable color. It can also produce chemiluminescence when reacting with substrates like luminol. HRP secondary antibodies are commonly used in immunoassays like ELISA, IHC, and western blots.

Alkaline phosphatase (AP): is an enzyme that produces a colorimetric signal from reacting with its substrate, pNPP. Alkaline phosphatase secondary antibodies can be used in immunoassays, IHC, and western blots.

Biotin: Biotin is a small vitamin molecule remarkable for its extremely strong binding affinity with avidin and streptavidin. Biotinylated secondary antibodies can contain multiple molecules of biotin, allowing for signal amplification or purification when combined with avidin/streptavidin conjugated to other reporters or beads.

Fluorescent labels: A number of commercial secondary antibodies are available conjugated with fluorophores that emit at virtually all colors of the visible spectrum. In choosing the fluorescent label, some considerations include: the specs and features of your imaging instrument, possible spectral overlap with other fluorescent-labeled antibodies, and any sources of natural autofluorescence within the sample. Secondary antibodies conjugated to fluorophores such as FITC, phycoerythrin (PE), allophycocyanin (APC), Alexa Fluor dyes, Texas Red, and tandem dyes, are common. Fluorescently-labeled secondary antibodies are widely used in fluorescent microscopy applications like immunofluorescence, immunohistochemistry, immunocytochemistry, as well as in multi-color Western blotting.


• 
  Check out our Fluorophores Poster to gain another perspective on how different fluorescent colors can be used in your experiments. This poster lists 100 commonly used fluorophores and plots their excitation and emission spectra on a two-dimensional map for a visual perspective.

Guidelines for choosing secondary antibodies

Pair the target and host species properly. As a general rule, the secondary antibody must be selective for the host species of the primary antibody. In addition, the host of the secondary must be a different species from the host of the primary. For example if the primary antibody is an IgG from mouse, then the secondary antibody should be an anti-mouse antibody from a species other than mouse.

The ideal secondary should also be selective to the isotype of the primary. For instance, for a mouse IgG primary, an anti-mouse IgG will work better than an anti-mouse IgM as a secondary antibody. If the primary antibody specifies an immunoglobulin subclass (such as IgG1, IgG2, IgG2a, etc.), then a secondary raised against that specific subclass may be more ideal. Antibody fragments such as Fab (antigen-binding fragment) region, Fc (constant fragment) are also suitable targets for secondary antibodies.

Anticipate potential cross-reactivities. Using multiple primary antibodies, especially with multiple host species, introduces risks of cross-reactivity, or unintended binding of secondary antibodies to targets aside from the designated primary. Ensure that all the primary antibody host species are raised in different species to avoid cross-labeling with the secondary antibody. Also check the supplier's datasheet for cross-reactivity information, as some secondary antibodies may inherently bind antibodies from other species. Some antibodies that are "cross-adsorbed" have undergone an additional purification step to further minimize cross-reactivity. These may be preferred in more sensitive applications.

Look for validation data. When using antibodies, it is always recommended to check for validation or testing data before committing to a purchase. Validation provides evidence that an antibody recognizes its specific target and works for a given application. For example, will a particular secondary antibody work well in immunofluorescence when looking at human tissue using a green fluorescent filter? Validation should include example data or figures from the application of interest. Read our brief antibody validation guide for more information.

Review product datasheets. Users should carefully examine the supplier-provided antibody datasheet, which contains a wealth of useful information such as validation tests, specifications, and protocols. Reviewing the protocol can help a user anticipate any potential incompatibilities with reagents, instruments, methods, or sample types. Species cross-reactivies are also often listed. Finally, citations to literature where the antibody have been used can help provide further insights into its reliability and applications.

References

• Saper, C. B. A Guide to the Perplexed on the Specificity of Antibodies. J Histochem Cytochem 57, 1–5 (2009). link
  
• Manning, C. F., Bundros, A. M. & Trimmer, J. S. Benefits and Pitfalls of Secondary Antibodies: Why Choosing the Right Secondary Is of Primary Importance. PLOS ONE 7, e38313 (2012). link
  
• Fung, P. & Dance, A. The Antibody Crisis: An Ongoing Discussion. Biocompare Editorial Articles (2017). link.