Why choosing the right secondary antibody matters?

Learn about the many factors that may affect the specificity, sensitivity and consistency of your secondary antibodies.

Secondary antibodies are commonly used as detection reagents in many different types of immunoassays such as western blotting, immunohistochemistry and immunocytochemistry or cellular imaging. However, the importance of selecting the right secondary antibody is sometimes overlooked.

Secondary antibodies, like any other antibody, are designed to target a specific antigen. All secondary antibodies target immunoglobulins (Igs) so they can bind primary antibodies and serve as detection reagents in  immunoassays. Thus, many of the factors that require consideration when selecting the appropriate primary antibody for your experiment, also apply to secondary antibodies. Selecting the right secondary for your assays can have a significant positive impact on your results. However, a poor secondary can have as much of a detrimental effect on your experiment as a poor performing primary antibody. Three parameters are mainly used to determine the performance of antibodies:

  • Specificity: refers to the degree with which the antibody detects its target antigen and not structurally similar antigens. This is particularly important for secondary antibodies as their target antigens are Igs, which homology is usually high between different types and even between species.
  • Sensitivity: refers to the amount of antigen that the antibody is able to detect. Sensitivity not only depends on the binding affinity of the secondary antibody (which is normally very high), the label and degree of labeling of the antibody also play an important role.
  • Consistency: refers to the ability of the antibody to perform with little variability when using different batches. Consistency will be mainly influenced by the source of the antibody and its clonality.

Performance of secondary antibodies is influenced by many different factors, for which the researcher can normally select from a range of different alternatives in order to ensure optimal results:

Host species
  • Goat and donkey: most popular host species. Commonly used to generate polyclonals.
  • Mouse and rat: commonly used to generate monoclonal secondary antibodies.
  • Others: rabbit, sheep, chicken, etc.
  • The host species to choose from is sometimes a personal preference. In our experience, there is no species specific difference in the quality of secondaries.
  • Using serum to block from the same host species of the secondary will significantly reduce background.
  • In general, all secondary antibodies should come from the same host species for multiple labeling.
  • Whole antibodies.
  • F(ab): fragment resulting from the digestion of the antibody with papain.
  • F(ab’)2: fragment resulting from the digestion of the antibody with pepsin.
  • Fragment antibodies are smaller and penetrate tissues more efficiently. This may be beneficial for certain applications such as immunohistochemistry.
  • Fragments reduce non-specific binding between Fc portions of the antibody and Fc receptors on cells. Useful in certain tissues such as spleen with high abundance of Fc receptors.
  • Enzymatic: HRP (more stable) and AP (more sensitive).
  • Fluorescent: Alexa Fluor® dyes, FITC, PE, APC, Cyanin dyes, etc.
  • Proteins: biotin.
  • Other: gold particles, magnetic beads, polymers.
  • The selection of the conjugate can greatly affect the sensitivity of the antibody.
  • Fluorescent conjugates vary in their brightness.
  • Biotin conjugates offer enhanced signal amplification. This is also true for enzymes or fluorochromes conjugated to polymers.
Target Protein (Igs)
  • Heavy chain: γ (IgG and subclasses IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4), μ (IgM), α (IgA and subclasses IgA1 and IgA2), ε (IgE), δ (IgD).
  • Light chains: κ (kappa), λ (lambda).
  • Igs from different species share similar structures. Antibodies against Igs from one species may cross-react with a number of other species unless they have been specifically adsorbed against the cross-reacting species.
  • The secondary antibody should match the class or subclass of the primary antibody used. This is especially true for monoclonal primary antibodies.
  • Subclass specific antibodies are intended for distinguishing between different subclasses of mouse IgG primary antibodies in multiple labeling experiments.
  • If the class and/or subclass of the primary antibody are not known, the anti-mouse IgG may be used since they recognize most of mouse IgG subclasses.
  • H&L: heavy and light chains (i.e. Fc and F(ab)/F(ab’)2).
  • Heavy or light chain: react with heavy chain only or light chain only.
  • Fc: react with constant region of heavy chain.
  • Hinge heavy chain: react with hinge region of heavy chain.
  • Alpha, delta, epsilon, gamma or mu chain: react only with the heavy chain of specific isotype.
  • F(ab) or F(ab’)2: react only with fragments.
  • Most polyclonal secondaries will target the heavy and light chains.
  • Secondaries targeting light chains will bind to any class or subclass as they share the light chains.
  • Heavy chain: γ (IgG and subclasses IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4), μ (IgM), α (IgA and subclasses IgA1 and IgA2), ε (IgE), δ (IgD).
  • Light chains: κ (kappa), λ (lambda).
In particular situations it may be good to consider the isotype of the secondary antibody itself. For instance, antibodies raised in mice which isotype is IgG2a will strongly bind to human leukocytes.
  • Polyclonals: bind several epitopes resulting in greater sensitivity, although it may be at the expense of specificity.
  • Monoclonal: detect a single epitope and result in highly specific antibodies, although it may be at the expense of sensitivity.
The benefits and limitations of clonality for primaries also apply to secondaries.
  • Affinity purified.
  • Pre-adsorbed: The solution containing secondary antibodies is passed through a column matrix containing immobilized serum proteins from potentially cross-reactive species. Non-specific secondary antibodies are retained in the column, while highly specific secondaries flow through. Example: a solution of secondary antibodies recognizing rabbit IgG is passed through a matrix containing immobilized sheep and bovine IgGs. Only antibodies highly specific to rabbit IgG will flow through the column, while secondary antibodies cross-reacting with sheep or bovine IgG will remain bound to the proteins in the column.
  • Most conjugated secondary antibodies are purified though affinity chromatography.
  • Pre-adsorbed (cross-adsorbed) secondary antibodies have gone through an additional purification step to increase their specificity and reduce risk of cross-reactivity.
  • Caution should be exercised when considering antibodies that have been adsorbed against closely-related species since they have greatly reduced epitope recognition and may recognize some monoclonals poorly.

Secondaries you can trust

We have generated a high quality range of well-defined secondaries to ensure the success of your experiments. Each of these secondaries has been extensively validated in key applications and quantitative data on their performance is available on their datasheets.

Our catalogue also includes a broad range of secondaries to ensure you find the product you need such as fragments or subclass-specific secondaries among many others.

Alexa Fluor® is a registered trademark of Life Technologies.  Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.

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