How to Choose a Secondary Antibody (2024)

In what species was the primary antibody developed?

Secondary antibodies are directed against the species of the primary antibody. Therefore, you will need a secondary antibody that is raised in a species different than the host species of the primary antibody. For example, if your primary antibody is raised in a mouse, you will need an anti-mouse secondary antibody raised in goat, rabbit, etc.

What is the class (isotype) and/or subclass of the primary antibody?

This question is primarily important when working with monoclonal antibodies. Polyclonal antibodies, however, are typically IgG class immunoglobulins. For this reason, the secondary antibodies will mainly be an anti-IgG antibody.

Monoclonal antibodies are most commonly developed in mice and occasionally in rats, hamsters, or rabbits. For example, if the primary monoclonal antibody is mouse IgM, one would want a secondary antibody that reacts with mouse IgM (anti-Mouse IgM).

If the primary monoclonal is one of the mouse IgG subclasses, almost any anti-mouse IgG secondary antibody should bind to it. If the subclass of the primary antibody is not known, then anti-Mouse IgG F(ab) secondary antibodies may be used since they recognize most mouse immunoglobulin subclasses.

There are many classes and subclasses of human and mouse IgG(s). Choosing a secondary may be difficult. However, one common factor among these IgG(s) are the light chains (kappa and lambda). In other words, IgG, IgM, IgA, IgD and IgE all have either kappa or lambda light chains. The heavy chain, however, is class specific.

Summary of Immunoglobulin Classes and Subclasses

• Immunglobulin Classes: IgG, IgM, IgA, IgE, IgD
• Human Immunoglobulin Subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2
• Mouse Immunoglobulin Subclasses: IgG1, IgG2a, IgG2b, IgG3
• Light Chains: Kappa, Lambda
• Heavy Chains: IgG (gamma), IgM (mu), IgA (alpha), IgD (delta), IgE (epsilon)

Additional Information on Antibody Reactivities

• Polyvalent: react with all classes
• Anti-Fc and heavy-chain specific: react with heavy chains only; therefore, they are class specific.
• Anti-Fab and whole molecule specific: react with heavy and light chains. Due to the light chain reactivity, they can react with all classes.
• Light chain (kappa, lambda) specific: react with all classes, since all classes use the same kappa or lambda light chains.

Do I use affinity purified antibody or an IgG fraction?

Most people prefer affinity isolated secondary antibodies because they provide the lowest amount of non-specific binding. However, in certain cases, IgG fractions may be considered, i.e. when the antigen of interest is rare or present in low abundance. In these situations high affinity antibodies are required. For example, during affinity isolation, some of the very high affinity antibodies may be removed because they bind so tightly to the affinity matrix that they are not able to be eluted.

What kind of label do I choose?

The label is very application dependent. For immunoblotting and ELISA, enzyme-labeled secondary antibodies are the most popular. Peroxidase is economical and a more stable enzyme, in general, than alkaline phosphatase. It has also become very popular for use in chemiluminescent detection systems. Alkaline phosphatase, on the other hand, is considered more sensitive than peroxidase particularly when colorimetric detection is used.

For cell or tissue staining, alkaline phosphatase, peroxidase, or secondary antibodies conjugated to a fluorochrome may be used. Common fluorochromes are FITC, phycoerythrin, CF™ Dyes, Atto Dyes, and Quantum Red. Associated applications are immunocytochemistry/immunofluorescence, flow cytometry, or immunohistochemistry.

To generate increased amplification, a two-step biotin/avidin system may be used. Biotin binds with very high affinity to avidin, resulting in an essentially irreversible interaction. A biotinylated secondary antibody is applied first, then avidin, ExtrAvidin, or streptavidin conjugated to an enzyme or fluorochrome binds to multiple sites on the biotinylated secondary antibody, thus amplifying the signal and resulting in greater sensitivity than that achieved with an antibody-enzyme or antibody-fluorochrome conjugate alone.

When should I use a pre-adsorbed secondary antibody?

Using pre-adsorbed antibodies will reduce non-specific background when working with tissues and cells. The process involves passing the secondary antibody over immobilized serum proteins from potentially cross reactive species. Therefore, if you are working with human tissues, choose a secondary antibody that has been adsorbed with human serum or human IgG.

When is a F(ab) or F(ab')₂fragment antibody necessary?

If working with tissues or cells that have Fc receptors, choose a F(ab) or F(ab')₂fragment when possible to eliminate non-specific binding. In more detail, F(ab')₂antibody fragments are used in assay systems where the presence of the Fc region may cause problems. Samples such as lymph nodes, spleen, and peripheral blood preparations contain cells with Fc receptors (macrophages, B lymphocytes, and natural killer cells), which could bind the Fc region of intact antibodies, causing high background staining. Use of F(ab')₂fragments ensures that any antibody binding observed is not due to Fc receptors.

How do I decipher the secondary antibody nomenclature?

• Anti-Goat IgG:Name of species antibody is directed against
• (H + L):Description of antibody specificity for heavy and light chains
• Highly cross-absorbed:Antibody has been absorbed to the serum of many different species
• CF™ 568 antibody:Description of the detection label
• Donkey:Host species of the secondary antibody