Secondary antibodies bind to a primary antibody once it has attached to its target antigen. They recognise the heavy chains of the primary antibody without interfering with primary–target binding, and are used chiefly for detection and purification.

Secondary antibodies usually carry a label or tag. By detecting that conjugate (or its by-product, e.g. the TMB product of an HRP reaction) you can quantify a protein, visualise it microscopically, or purify it from a complex solution.

Species

A secondary must be raised in a species different from both the primary antibody and the sample, to avoid cross-reactivity. For a mouse sample with a goat primary, for example, an anti-goat secondary raised in another species (e.g. rabbit) is used.

Isotype

IgG is the most common isotype, as IgG molecules from different species bind one another well. Subclasses include IgG1, IgG2a/b/c, IgG3 and IgG4; IgA, IgD, IgE and IgM are used less often.

Conjugate

The conjugate is chosen for the application — enzymatic tags (HRP, AP) for ELISA/western blot colour development, or fluorophores (FITC, AMCA, Rhodamine, Cy3) for immunofluorescence and IHC. Common conjugates:

• Horseradish peroxidase (HRP) — enzyme
• Alkaline phosphatase (AP) — enzyme
• Biotin
• FITC — fluorophore
• AMCA — fluorophore
• Rhodamine — fluorophore
• Cy3 — fluorophore