Antibody validation

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​​​​​​​​​​​​​A. Background & Definitions

Currently, there are no official quality standards for commercially available antibodies. The robustness and reproducibility of antibody-based methods critically depend on the specificity and sensitivity of antibodies used - with respect to intended application. Therefore, validation steps are warranted for each new antibody under the conditions used in the specific laboratory environment.

B. Guidance & Expectations

  • The International Working Group on Antibody Validation (IWGAV) suggests five conceptual pillars for validation of antibodies:
    • Genetic strategies: The expression of the target protein is eliminated or significantly reduced by genome editing or RNA interference,
    • Orthogonal strategies: Expression of the target protein is compared with an antibody-independent method,
    • Independent antibody strategies: Expression of the target protein is compared using two antibodies with nonoverlapping epitopes
    • Expression of tagged proteins: The target protein is expressed using a tag, preferably expressed at endogenous levels
    • Immunocapture followed by mass spectrometry: The target protein is captured using an antibody and analyzed using MS
  • At least one of the above listed strategies should be used as a minimum criterion for claiming that a particular antibody has been adequately validated for a specific application (further information can be found here: Uhlen et al. Nat Methods. 2016;13:823-7).
  • Authenticate antibodies used by finding or generating stable unique identifiers: Research Resource Identifiers (#RRID) are ID numbers assigned to help researchers cite key resources in the biomedical literature to improve transparency of research methods (https://www.rrids.org/).​


Extra care has to be taken:

  • Antibodies need to be validated for the intendent use. For example, an antibody validated for an unfolded condition (Western blotting) may not work in native context assays (immunohistochemistry or immunoprecipitation), and vice versa.
  • Lot variation can be a concern, particularly for polyclonal antibodies and particularly when raised against an entire protein and undefined epitope.
  • Don't rely only on the manufacturers' data sheet. Test/validation results shown on the product sheet will no longer be representative after the batch or lot has been replaced by its successor, unless the data have been reproduced with the new batch/lot.

C. Resources

  • Information-sharing requirements [1]
  • High-quality antibody databases [2]
  • International frameworks for antibody validation standards:
    • The European Monoclonal Antibody Network (EuroMAbNet): Roncador G, Engel P, Maestre L, Anderson AP, Cordell JL, et al. The European antibody network's practical guide to finding and validating suitable antibodies for research. MAbs. 2016; 8:27-36
    • The International Working Group on Antibody Validation (IWGAV): Uhlen M, Bandrowski A, Carr S, Edwards A, Ellenberg J, et al. A proposal for validation of antibodies. Nat Methods. 2016;13:823-7.
  • RRID portal [3]

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