• What are secondary antibodies?
• How to choose a secondary antibody
• Browse our secondary antibodies
• Why use secondary antibodies?
• Types of secondary antibody
• Choose the right conjugate
• Secondary Antibodies applications
• Cross-reactivity, background and how to avoid it
• Frequently asked questions

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.

Choosing a secondary antibody is a process of elimination. Five properties have to match your experiment, and getting any one of them wrong is the usual cause of weak signal or high background. Work through them in this order.

1. Match the host species of your primary. The secondary must be raised against the species your primary antibody came from. A rabbit primary needs an anti-rabbit secondary; a mouse primary needs an anti-mouse secondary. This is the single most common mistake — check the primary's datasheet rather than assuming.
2. Check the secondary's own host against your sample. The secondary must be raised in a species that is not present in your sample, or it will bind endogenous immunoglobulin. Staining mouse tissue with a mouse primary is the classic trap; it needs either a mouse-on-mouse detection system or a different primary.
3. Pick the conjugate for your readout. Enzymes (HRP, AP) for blots and plate assays; fluorophores (FITC, Cy3, Alexa-type dyes) for imaging and flow cytometry. Unconjugated secondaries are for building your own detection system or for biotin–streptavidin amplification.
4. Decide on the fragment and specificity. Whole IgG (H+L) is the default and gives the strongest signal. F(ab')₂ fragments remove the Fc region, which matters when your sample has Fc receptors — immune cells, spleen, whole blood. Fc-fragment-specific and heavy-chain-specific secondaries reduce binding to light chains.
5. Decide whether you need cross-adsorption. If you are multiplexing, or your sample contains immunoglobulin from a related species, a cross-adsorbed (pre-adsorbed) secondary has been passed over other species' IgG to strip the antibodies that would bind them. It costs more and gives slightly lower signal, but it is the difference between a clean multiplex and an uninterpretable one.

If you are still unsure after those five, the deciding factor is usually the application — a secondary that performs well on a western blot is not necessarily the right choice for immunofluorescence. The application notes below cover the differences.

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

Whole IgG, F(ab')₂ and Fab fragments

A whole IgG secondary carries both the antigen-binding arms and the Fc stem. It is the default choice and gives the highest signal per molecule. Digesting away the Fc leaves an F(ab')₂ fragment — still divalent, still able to bind two primary molecules, but no longer recognised by Fc receptors. Use F(ab')₂ when working with immune cells, spleen, blood or any tissue where Fc receptor binding produces background. A monovalent Fab fragment is smaller again and penetrates dense tissue better, at the cost of signal.

H+L, Fc-specific and heavy-chain specific

An anti-IgG (H+L) secondary is raised against both the heavy and light chains. Because light chains are shared across immunoglobulin classes, an H+L reagent will also detect IgM and IgA from the same species — usually helpful, occasionally not. An Fc-fragment-specific secondary binds only the heavy-chain constant region, so it will not pick up light chains or other isotypes. Choose Fc-specific when you need to detect IgG and nothing else.

Polyclonal and monoclonal secondaries

Almost all secondary antibodies are polyclonal, and deliberately so: a polyclonal population binds multiple epitopes on each primary molecule, which is exactly the signal amplification you want. Monoclonal secondaries exist for applications that need absolute lot-to-lot consistency, but they give lower signal for the same reason.

Conjugated and unconjugated

Most secondaries arrive pre-conjugated to an enzyme or fluorophore. Unconjugated secondaries are used when you are building a custom detection system, labelling in-house, or working with a biotin–streptavidin amplification chain where the extra layer buys sensitivity.