Secondary antibodies are staple tools in immunological applications for detecting, quantifying, or purifying target proteins and other molecules.
While the primary antibody is specific to the target molecule, the ideal secondary antibody should bind specifically to the primary antibody, determined largely by the latter’s host species and antibody class or isotype (IgG, IgA, IgD, IgE, and IgM).
In addition, the secondary antibody is often conjugated to another molecule for detection, such as to a fluorophore, biotin, horseradish peroxidase, or alkaline phosphatase.
The diversity of different target species and conjugates allow for a variety of possible combinations, providing flexibility in designing experiments.
Listed below are some general guidelines in selecting the appropriate secondary antibody for your experiments.
Selectivity to the primary antibody:
As a general rule, the secondary antibody must be selective for the host species of the primary antibody.
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.
If using multiple primaries, ensure that they are raised in different species to avoid cross-labeling with the secondary antibody.
Detection method:
Different kinds of labels can be covalently attached to secondary antibodies for their detection.
Depending on the experimental application, choices of labels include enzymes, biotin, and fluorophores.
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 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.
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Secondary antibodies conjugated to
green-,
red-,
orange-, or
blue- emitting fluorophores such as FITC, phycoerythrin (PE), TRITC, and AMCA, respectively, are common.
These kinds of antibodies are widely used in applications such as in ELISA, immunofluorescence, flow cytometry, and immunocytochemistry.
Download our Fluorophores Poster containing a list of 100 commonly used fluorophores to gain a bigger perspective on how different fluorescent colors can be used in your experiments.
Application and validation:
Antibody companies may have successfully tested or validated their antibodies on certain applications.
It is useful to check the supplier product pages for such information, as that may save you considerable time and money.
A given antibody may also have been reviewed by another user.
Check our listing of
user-written product reviews
to see how well the antibody may have worked for others, anticipate any potential shortcomings, or even learn procedural tips.
Relevant Literature:
- Saper, C. B. A Guide to the Perplexed on the Specificity of Antibodies. J Histochem Cytochem 57, 1–5 (2009).
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- 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).
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- Fung, P. & Dance, A. The Antibody Crisis: An Ongoing Discussion. Biocompare Editorial Articles (2017).
link.