Flow Cytometry (FCM) /FACS | Secondary Antibody

Antibody Staining

Direct antibody staining utilize labeled primary antibody as one-step staining. It is more convenient for multiplex targets staining.

Indirect antibody staining for flow cytometry is a kind of indirect immunoassay, which the secondary antibodies were needed as detector antibodies. Secondary antibodies are the "anti-antibodies" against the primary antibody you are using. For example, if your primary is a mouse monoclonal, you will require an anti-mouse secondary. The secondary antibody was conjugated with a variety of fluorochromes or chromogens.

The advantage of indirect immunoassay:

• High sensitivity: More than one labeled antibody is bound per antigen molecule; The signal intensity could be amplified on a single epitope.
• Flexible: Different primary antibodies can be used with a single labeled secondary antibody; The reservoir of primary antibodies suitable for flow cytometry would be broadened for no limitation of fluorochrome labeling.
• Cost-saving: Fewer labeled antibodies are required.

The disadvantage of indirect immunoassay:

• Complexity: In multiplex staining experiments, choosing for appropriate secondary antibody could be complex and time consuming.
• Species cross-reactivity: Secondary antibodies may cross-react with species other than the target. The use of pre-adsorbed secondary antibodies can prevent cross-reactivity.
• Background: Samples with endogenous immunoglobulins may exhibit a high background with indirect methods.

How to choose a secondary antibody:

Host species Class/ subclass of antibody Human immunoglobulin isotypes
The secondary antibody is raised against the host species used to generate the primary antibody, for instance, if you use a primary antibody raised in mouse, you will need an anti-mouse secondary antibody raised in a host species other than mouse (e.g. goat anti-mouse secondary). The secondary antibody has to be directed against the isotype of the primary antibody.

Polyclonal primary antibodies are generally raised in rabbit, goat, sheep or donkey and are generally IgG isotypes. The secondary antibody therefore, will typically be an anti-IgG H&L (Heavy & Light chains) antibody.

Monoclonal primary antibodies are commonly raised in mouse, rabbit and rat. For example, if the primary monoclonal antibody is a mouse IgG1, you will need an anti-mouse IgG.
Classes or isotypes: IgG (γ heavy chains), IgM (μ), IgA (α), IgE (ε), IgD (δ)

Subclasses: IgG1 (γ1 heavy chains), IgG2 (γ2), IgG3 (γ3), IgG4 (γ4); IgA1 (α1), IgA2 (α2)

Types: κ light chain, λ light chain

Subtypes: λ1, λ2, λ3, λ4