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By Colin O'Neill, 2018 Novus Intern
The specificity and affinity of an antibody for its target protein—and only its target is valued as an indicator of its experimental reliability. The frustrating ability of an antibody to bind to multiple proteins has prompted a reproducibility crisis in the life science community. Specifically, if an antibody varies in specificity between batches, attempts to reproduce significant findings with that antibody may fail. With samples, time, and funding at stake, scientists have developed novel validation techniques to ensure antibodies solely bind to their intended targets to avoid false positives and ambiguous results. Nature's 2015 article "Reproducibility Crisis: Blame it on the Antibodies" by Baker outlined this issue 1.
The reproducibility crisis was popularized by Yale pathologist Dr. David Rimm MD while exploring tumor diagnostics. After a failed antibody staining experiment inspired him to host educational seminars on the importance of antibody validation, he began a lecture circuit and webinar series promoting KO validation, in hopes that others may learn from his misfortune. In response to increasing concern within the scientific community, the Global Biological Standards Institute (GBSI) and International Working Group for Antibody Validation (IWGAV) have set guidelines to standardize antibody validation, now known as "The Five Pillars of Antibody Validation".
These 5 pillars consist of genetic strategy validation, independent antibody validation, orthogonal validation, expression of tagged proteins validation, and biological strategies validation. The biological strategies validation utilizes biological and chemical modulation of protein expression to demonstrate antibody specificity to the target protein. Independent antibody validation uses data from several antibodies targeting different epitopes within the same protein, and specificity is assessed by consistency in detection between different antibodies.
Genetic strategy validation, as exemplified by genetic knockout (KO) is the most reliable of these pillars. In KO validation, a negative control can be produced using CRISPR/Cas9 induced deletion of the target-encoding gene. A wildtype (WT) sample containing the target protein is compared with the KO control to qualitatively assess specificity, indicated by the absence of binding in the negative control. Orthogonal, independent, tagged expression and biological strategies validation techniques are valid, but less frequently used alternatives.
A negative control can be produced with a temporary knockdown (KD) approach using siRNA or shRNA but a CRISPR/Cas9 KO control is preferred as it provides a permanent and stable complete gene knockout. It is worth noting that alternatives to CRISPR/Cas9 KO exist. Zinc finger nucleases (ZFN) and transcript or activator-like effector nucleases (TALEN) are both enzymes with the ability to perform gene KO. ZFN and TALEN’s inferior targeting efficiency and complicated nuclease design have prevented researchers from regularly using these increasingly antiquated alternatives 2.
While KO validation allows investigators to determine the specificity of an antibody for its target protein, antibodies must also be validated for a specific application. For example, the affinity of antibodies in fixed tissues differs from non-fixed tissue due to antigen denaturation. KO validation alone will not account for this potential conformational variance of the antigen 3.
Although antigen denaturation poses challenges for antibody affinity, antibodies themselves may denature throughout the shipping process as well. The slightest variance from native conformation may impact specificity greatly, prompting many researchers to perform independent validations using KO lysates. Extreme precautions are taken to eliminate and prevent denaturation of Novus products, and all products are covered by our 100% Guarantee.
Learn more about Novus' Antibody Validation Efforts
Colin O'Neill is a student at the College of Veterinary Medicine
and Biomedical Sciences, Colorado State University, Fort Collins, CO.
Colin is completing undergraduate research in molecular neuroscience
and Alzheimer's pathways.
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