Commercial antibodies frequently lack validation data and only 30-50% have demonstrated specificity for their targets. Antibodies have been shown to detect proteins of incorrect molecular weight, no protein at all, or worst, detection of a protein of the correct molecular weight which is not the intended target. Furthermore, the correlation in the protein binding of the same antibody from different lots can be as low as R2=0.038. The program director of the Human Protein Atlas consortium, Matthias Ulhen, said in 2014 that after testing more than 25,000 antibodies from 50 plus suppliers the vast majority did not function as intended.
Lack of confidence in antibody reproducibility has many research consequences such as false conclusions, uninterpretable or misinterpreted results, wasted samples, inability to replicate published results, paper retractions, stronger reviewers’ challenges, and researchers’ time and money wasted. Commercial antibodies are often screened and optimized for narrow experimental conditions and may not work as advertised causing misleading or irreproducible results. One solution to bring confidence to your results is to validate each antibody for your own research needs.
What is testing vs validation?
There is a difference between testing an antibody and validating it. In testing, a positive result is the acceptance criteria, but the signal must be in the right place and in an appropriate sample. Validation goes beyond a positive test signal, to examine the boundaries/truth of the positive signal. Truly validating an antibody determines whether the reagent is specific, selective, and reproducible and under what conditions. Validation is also different from conformity, which implies that the antibody should perform as described by the provider (i.e., when using the same cell line including treatment, protein preparation, dilution, technique, and detection).
To validate an antibody, it must be at maximal dilution and:
- Bind the intended protein (specificity),
- React minimally with other proteins (selectivity), and
- Consistently and repeatedly exhibit the same result (reproducibility).
It means a very good signal in the correct sample and hardly any signal in a negative control (used at the same dilution). Because antibody validation demonstrates that the performance characteristics are suitable for its intended use with specific laboratory research tools, this means there is no validation strategy that serves as a gold standard to assess antibodies across all applications, since the expected performance in one application does not necessarily predict applicability in another one. Therefore, each researcher should validate an antibody in the specific assay and biological sample of interest, using appropriate loading controls and negative controls based on cell or tissue-specific expression.
Is there a link between QC standards and reproducibility, and why should I worry about it?
Antibodies are the only extensively used reagent in molecular biology that are uncharacterized at the molecular level and hard to validate by users. Similar to the qPCR guidelines (MIQE guidelines), the implementation of the Western-blot minimal reporting standards (WBMRS) has been proposed to improve Western-blot reproducibility.
Under these guidelines, the minimum information needed to reproduce antibody results are:
- Antibody provider catalog and lot numbers
- Specific experimental design
- Sample information
- Antibody usage conditions
To replicate published results and obtain reliable results, antibody validation regarding specificity, selectivity and reproducibility must be carried out using the same experimental setup. Unfortunately, the conditions used to validate a particular cell line or tissue type cannot necessarily be used to prove the antibody is functional in a different one. Additionally, reagent aging, instability, inappropriate storage conditions and handling, incorrect dilution, and host of other factors can also lead to low performance. Retraction Watch collates and reports a substantial number of papers retracted, and academic reputations damaged as result of poor Western-blot practices.
How can I make sure my antibody is working correctly?
To set up standards and implement the creation of a catalog of validated antibodies, different researchers have participated in initiatives such as Human Antibody Initiative (HAI) and International Working Group for Antibody Validation (IWGAV). These groups have defined the following 5 criteria for antibody validation in an application- and experimental condition- specific manner:
- Genetic strategies using tissues with differential protein expression, and/or exogenous: overexpression, reduction (knockdown), or elimination (knock out) of the target protein. This requires the use of more than one reagent (eg: at least 2 different siRNA molecules) or negative cell lines, and includes the verification by RT-qPCR to establish a direct link between mRNA reduction and protein detection by the antibody.
- Orthogonal strategies carrying out protein quantification in diverse cell samples using an antibody-independent approach such as mass spectrometry-based targeted proteomics and comparing it with the antibody signal.
- Expression of tagged proteins that enable parallel detection. For example, affinity tags such as FLAG or fluorescence proteins like green fluorescent protein should show a similar positive signal that is comparable to the specific antibody being validated. If these results greatly conflict, this means the antibody is cross-reactive with other proteins or epitopes.
- The independent antibody strategy tests at least 2 different antibodies with non-overlapping epitopes across a panel of different sample types (e.g., cell, tissue); this minimizes the chance of having the same off-target binding signals. Ideally, samples with variable protein expression should be used, including those with knockdown and knockout of the gene being targeted by the antibody.
- Immunocapture followed by mass spectrometry (IMS) is best for finding off-targets. In this strategy, proteins are isolated from solution after binding to the target antibody and then they are identified by MS. The antibody is considered specific if the three top peptides are derived from the expected target.
At a minimum, one of the above strategies must be applied to validate any antibody, but more is better. It may not be feasible to perform every strategy, but genetic and independent antibodies strategies are an economical place to start. Taking the step to validate your antibodies brings greater quality and accuracy to your research that is repeatable, which means higher impact work that benefits the entire research community.
Author: Johanna De Castro Arce, Ph.D. Field Application Scientist
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