Science Forum: Antibody characterization is critical to enhance reproducibility in biomedical research
- Department of Biochemistry, Emory University School of Medicine, United States
- Department of Respiratory Sciences, University of Leicester, United Kingdom
- Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Canada
- The Development Studies Hybridoma Databank, University of Iowa, United States
- The Michael J Fox Foundation for Parkinson’s Research, United States
- Institute for Protein Innovation, United States
- Department of Neurology, Emory University School of Medicine, United States
- Department of Neurology and Program in Neuroscience, Harvard Medical School and Beth Israel Deaconess Medical Center, United States
- Center for Open Science, United States
- Department of Microbiology and Immunology, Stanford University School of Medicine, United States
- Department of Neuroscience, University of California, San Diego, United States
- Department of Physiology and Membrane Biology, University of California, Davis School of Medicine, United States
- Addgene, United States
- Frederick National Laboratory for Cancer Research, United States
- Specifica, a Q2 company, United States
- Department of Research and Development, Abcam, United Kingdom
- CiteAb, United Kingdom
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Science Forum: Antibody characterization is critical to enhance reproducibility in biomedical researcheLife 13:e100211.https://doi.org/10.7554/eLife.100211 - Cite
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Figures
Figure 1
Key projects related to antibody characterization.
Timeline showing when the projects discussed in this article were started: the Protein Capture Reagents Program and the Affinomics project were funded for five years (as indicated by dashed lines). …
Tables
Table 1
The ‘five pillars’ of antibody characterization.
In 2016 an ad hoc International Working Group for Antibody Validation introduced the five pillars of antibody validation/characterization: (i) genetic strategies; (ii) orthogonal strategies; (iii) …
| Pillar/strategy | Description | Specificity | Example applications | Pitfalls | |
|---|---|---|---|---|---|
| i | Genetic strategies | Knock-out/ knock-down target gene | High | WB, IHC, IF, ELISA, IP | Requires a genetically tractable system and awareness of potential confounders (such as alternative isoforms) |
| ii | Orthogonal strategies | Compare results from Ab-dependent and Ab-independent experiments | Varies | WB, IHC, IF, ELISA | Requires variable expression of the target and cannot entirely rule out non-specific binding to similar proteins |
| iii | Independent antibody strategies | Compare results from experiments using unique Abs to the same target | Medium | WB, IHC, IF, ELISA, IP | Requires the purchase of multiple Abs and knowledge of their epitopes |
| iv | Recombinant strategies | Experimentally increase target protein expression | Medium | WB, IHC, IF | Overexpression of exogenous protein can lead to overconfidence in the specificity of the Ab |
| v | Capture MS strategies | Use MS to identify protein captured by Ab | Low | IP | Requires access to MS and it can be challenging to distinguish between Ab binding target vs protein bound to target |
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Ab: antibody; ELISA: enzyme-linked immunosorbent assay; IF: immunofluorescence; IHC: immunohistochemistry; IP: immunoprecipitation; MS: mass spectrometry; WB: Western blotting.
