Antibody valitation: Practical guide to finding and validating suitable antibodies for research
2.C Review product information
You should review the product data sheet from the commercial antibody provider or the scientific literature to obtain the following relevant information.
1. Antigen used for antibody production: The antigen used as an immunogen dictates the range of available epitopes and can help predict the specificity of your antibody for the target protein. If this information is unavailable you should contact the company and ask if they will supply this data. If you want antibodies to a specific region in the target protein or you wish to avoid a region because it shares sequence identity with a related proteins then companies may be willing to tell you if this region is contained within their immunogen even if they refuse to disclose the actual immunogen. If no information is available, you have to proceed on the basis that the antibody may bind anywhere within the full-length protein. Be aware that many antibody suppliers do not test antibodies for their potential cross-reactivity with related proteins.
2. Monoclonal versus Polyclonal: It is important when planning a prolonged project to ensure there you can obtain sufficient antibody to complete the study. Particularly with polyclonal antibodies it may be worth testing the reagent and then buying several vials from the same batch to avoid problems with reproducibility.
3. Clone name or product code: This enables you to identify the antibody in the scientific literature and avoid buying the same antibody from different suppliers. This is generally much easier for monoclonal antibodies, which should have a defined clone name, than for polyclonal antibodies.
4. Isotype: Different isotypes may be useful for experiments using multiple antibodies, e.g. enabling detection using isotype specific secondary reagents. The antibody isotype also affects an antibody’s ability to engage with complement or FcR present in cells of the innate immune system, which is of critical functional relevance for therapeutic reagents.
5. Host species: This information can help distinguish individual antibodies and is also relevant for multiple labelling experiments. Using same species antibodies and tissues (e.g. a mouse monoclonal antibody to stain mouse tissue) needs additional strategies to avoid secondary antibodies binding endogenous immunoglobulins, a situation that can be avoided when the antibody is raised in a different species.
6. Species reactivity: An antibody is usually raised to a protein from a single species, commonly human or mouse. The antibody may recognise the orthologous antigen from another species if the epitope(s) is sufficiently similar. It is important to be aware of the difference between experimentally determined species cross-reactivity data versus predictions based on sequence identity. If reactivity with different species is not mentioned, this may mean that other species have not been tested. If in any doubt ask the supplier for further information.
7. Tested applications: Antibody datasheets should list the techniques that have been tested and found to work with the antibody. There may also be some further technical guidance and suggested working dilutions. An illustrated example of the successful use in any of these applications should be provided, as this is necessary to critically judge the quality of the data that has been obtained and the conclusions that have been drawn. An antibody may be judged as unsuitable for blotting because it detects multiple bands, but these may be isoforms of the same protein. Be aware that if an application is not listed, this may not mean that the antibody is unsuitable. It may simply mean that it has not been tested and it is unknown how the antibody will perform. Many suppliers will, however, specify that an antibody is not recommended for a particular technique if it has been tested and does not work.
- Step 1: Define your initial requirements
- 1.A Identify the target antigen
- 1.B Define antibody requirements
- 1.C Decide on the experimental techniques you wish to use
- Example step 1
- Step2: Collect information and find existing antibodies
- 2.A Review the published peer-reviewed literature
- 2.B Find existing antibodies
- 2.C Review product information
- 2.D Match antibody data to existing information
- 2.E Study the individual company catalogues
- 2.F Accurately identify individual antibodies
- Example step 2
- Step 3: Assess existing validation data and decide whether further validation is necessary
- 3.A Reactivity with the target antigen
- 3.B Its suitability for use in your intended applications
- 3.C Decide whether further validation is necessary
- Example step 3
- Step 4: Reproducibility and dissemination of data
- Positive and negative controls for antibody validation
- Articles about antibody validation