Secondary Antibodies
Secondary antibodies have several functions in applications. They offer a route to label a wide range of primary antibodies with a hapten without having to label the primary antibody, which is usually comparatively expensive, and any mistakes could result in the loss of, or loss of function of, the primary antibody.
The use of a secondary antibody can also help to increase sensitivity of a method, as several antibodies can bind to the primary, each carrying more reporter molecules.
Selecting a secondary antibody
The secondary antibody should be raised against the species used as the host for the primary antibody. Consideration if the primary antibody isotype or format may be necessary. Whilst general secondaries raised against IgG (H+L) will recognise a range of sites on the primary antibody, this can allow unwanted cross reactivity. Where a secondary is raised against a specific isotype, this may offer technical options and advantages, but extra care may be needed at the bench to ensure the secondary specificity and primary antibody isotype are compatible.
Conjugates
Popular conjugates include:
- Haptens, such as biotin or click chemistry motifs, that can be easily reacted with another molecule at a later stage to form a complex,
- Enzymes, including peroxidase or alkaline phosphatase, that can be reacted with a substrate to enable visualisation
- Fluorophores, such as DyLight or AZ Dyes for fluorescent detection.
