Detect gene expression, knockdown, or knockout with confidence
Only 30-50% of commercial antibodies demonstrate specificity for their targets. Furthermore, the correlation in the protein binding of the same antibody from different lots can be as low as r2=0.038. 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. 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. There is an interesting simile described in Bordeaux, et al.; when buying a DNA isolation kit, there is generally the security it will isolate DNA and not proteins, but the same is not true for antibodies.
The antibody crisis has many 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.
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 in a relevant sample. Validation goes beyond a positive signal, it tests the boundaries/truth of the positive signal. Validation is testing that the reagent is specific, selective and reproducible. To validate, the reagent must be at maximal dilution:
It means a very good signal in the correct sample and hardly any signal in a negative control using the same dilution. Validation is also different from conformity, the latter implies that the antibody should perform as described by the provider (same cell line including treatment, the protein preparation, dilution, technique, and detection). Validation can be defined as the demonstration, using specific laboratory research tools, that the performance characteristics of any analytic reagent are suitable for its intended use. There is no gold standard validation strategy to assess antibodies across all applications because expected performance in one application does not necessarily predict applicability in another one. Therefore, each researcher should validate the analyte in the specific assay and biological sample of interest, using appropriate loading controls, and negative controls based on cell or tissue-specific expression.
Antibodies are the only extensively used reagent in molecular biology 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. Retraction Watch reports a substantial amount of papers retracted, and academic reputations damaged as result of poor Western-blot practices.
To reproduce antibody results there is a minimal information needed:
However, in order to replicate published results and obtain reliable results, antibody validation regarding specificity, selectivity and reproducibility must be carried out for the singular experimental setup. The validation data using a particular cell line or tissue cannot necessarily be used to prove the antibody to be functional in a different one. Additionally, reagent aging, instability, inappropriate storage conditions and handling, incorrect dilution, among other factors can also lead to low performance.
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 5 criteria for antibody validation in an application- and experimental condition- specific manner:
Minimum one strategy of validation must be applied, and although some of them are not at hand in all laboratories, genetic and independent antibodies strategies can be applied without incurring in excessive investments, and certainly, the benefit at long run is higher, regarding the quality, and accuracy, of the results and the derived conclusions.
Author: Johanna De Castro Arce, Ph.D. Field Application Scientist