Antibody validation guidelines released
5 Sep 2016 by Evoluted New Media
An international working group have published the first proposals for validating antibody specificity.
An international working group have published the first proposals for validating antibody specificity.
Published today in Nature Methods, the proposals by the International Working Group on Antibody Validation (IWGAV) are intended to address needs for antibody functionality and reproducibility.Dr Mathias Uhlén, professor of microbiology and IGWAV chair said: “This publication is an important first step towards the development of widely accepted standards for validating antibodies and ensuring high quality and consistent antibodies for biomedical research.
“We look forward to receiving feedback from the broader community of antibody users, publishers, funding agencies and producers to help strengthen this initial proposal and ensure the reliability of these essential tools of biomedical research.”
The group has identified five pillars to help guide antibody validation for research. They are:
- Genetic strategies: Measure the relevant signal in control cells or tissues in which the target gene has been knocked out or knocked down using techniques such as CRISPR/Cas or RNAi.
- Orthogonal strategies: Use an antibody-independent method for quantification across multitudes of samples and then examine the correlation between the antibody-based and antibody-independent quantifications.
- Independent antibody strategies: Use two or more independent antibodies that recognise different epitopes on the target protein and confirm specificity via comparative and quantitative analyses.
- Expression of tagged proteins: Modify the endogenous target gene to add sequences for an affinity tag or a fluorescent protein. The signal from the tagged protein can be correlated with detection through antibody-based methods.
- Immunocapture followed by mass spectrometry (MS): Couple immunocapture, the technique of isolating a protein from a solution through binding with a target-specific antibody, with MS analysis to identify proteins that interact directly with the purified antibody as well as proteins that may form a complex with the target protein.
Although each individual pillar could provide evidence of specificity, the IWGAV recommends the use of multiple pillars to confirm antibody validation.
The IWGAV was established in 2015 with operational support provided by Thermo Fisher Scientific.