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Antibody validation Practical guide to finding and validating suitable antibodies for research

1.B Define antibody requirements

The following information will help you decide which type of antibody may be the best choice for your application.

  • 1. Polyclonal versus monoclonal: This depends upon the availability of the antibody. Decide whether the technical advantages of having multiple epitopes recognised by a polyclonal antibody which may give stronger labelling or work in a wider range of techniques is important. This must be weighed against the greater risk of off target cross-reactivity, batch-to-batch variability and possibly limited availability compared to monoclonal antibodies, which are specific for a single epitope and can be produced in unlimited quantities.
  • 2. Isotype and host species: This may be important when you are interested in the detection of multiple antigens in the same samples using multiple antibodies. In this case you should select primary antibodies that have been raised in different species or that possess different isotypes. Accordingly, these antibodies can be visualised with differently labelled species–specific or isotype-specific secondary antibodies. Also, to avoid cross-reactivity of the secondary anti-immunoglobulin antibody, the species of the primary antibody should be different from the species of your sample. This will avoid the detection of endogenous immunoglobulins. For example, if you are studying a mouse protein you should choose a primary antibody produced in rabbit, goat or rat.