Research Antibody Validation – Industry’s Perspective
The aim of this document is to help researchers understand what constitutes “research antibody validation” from an industry perspective. Please note, this document refers to research antibodies used in pre-clinical studies and not therapeutic antibodies.
Issues with commercially-available antibodies
Commercially-available antibodies have become a key tool in biomedical research, used in a spectrum of experiments from measuring protein expression to purifying gene products. However, these antibodies, whether they are monoclonal or polyclonal, can be of a substandard quality. They can:
- Bind to a different target than advertised, or to more than one target (cross-reactivity, non-specificity)
- Be non-optimally formulated – see below
- Give high levels of aggregation or degradation
- Not be suitable for their advertised application, g. fluorescence-activated cell sorting (FACS) versus Western blot (WB) – see below
- Not be at the concentration reported
The sum of the above can mislead research with false positive or negative results.
This failure is fuelled by a number of factors, including:
- Questionable antibody validation methods
- A lack of clear quality standards
- Opaque supply chains – often the identical antibody is sold by multiple companies
- Manufacturers failing to validate their products
- Insufficient research funding for scientists to validate purchased antibodies and/or insufficient research funds to confirm results with independent antibody preparations.
Poor quality antibodies compromise the accuracy and validity of results, further contributing to the ‘reproducibility crisis’ and the waste in resources and reagents.
Like any reagent used in experimentation, antibodies must undergo quality control (QC) prior to use.
Ideally, research antibodies should be formulated in PBS, which facilitates their usage in a range of applications (e.g. labelling, WB, IHC, flow). This is also an indication that the antibody is stable. Some antibodies are supplied with additives (Tris, Proclin, Sodium azide etc.) which may adversely affect some applications. For example, excess protein in the buffer will lead to poor biotinylation. If the researcher is not aware, performs biotinylation, does not assess its level, uses it and gets a negative result, this could be a false negative. Similarly, sodium azide can be toxic to cells.
In addition, the following criteria must be met in the validation of antibodies:
- The antibody needs to be identifiable – this could include purpose/application, a clone number, commercial product number, antibody ID, whether it is monoclonal or polyclonal, or even the amino acid sequence
- The target antigen needs to be defined, whether it be in the form of the chemical formula of the target, the corresponding gene or a cell type/tissue. Importantly, the version of the target recognised by the antibody – whether it is in the native form, or denatured, or even chemically modified should also be noted
- Assessment of Cross-reactivity (CR) is needed to determine the usability of an antibody in a specific context. For example, an antibody should only identify a single protein of the correct size on a western blot, not multiple protein bands. While specificity can be confirmed on a western blot, the demonstration of specificity in immunohistochemical or flow cytometry experiments can be much more challenging, as the specific protein-target can be difficult to confirm. It may even be important to ensure that reagents used in this assessment are also “mono-specific” – demonstrating lack of CR can be difficult and data easily misinterpreted. The ideal and perhaps most straightforward way to assess specificity of an antibody is to create an isogenic cell-line through over-expression or genetic deletion of the target of interest.
- Due to the difficulty of determining antibody concentrations, supplied data on product labels must be viewed as a preliminary estimate and scientists should determine the optimal concentration of active antibody for each assay under their own conditions
- Binding strength (affinity constant or binding constant) information can be used as an indication of an antibody’s performance. Antibodies with low affinity often lead to weak signals, nonspecific background binding, and increased costs due to the high concentrations and thus, volumes, needed for experiments. High-affinity antibodies are preferable for nearly all applications
- The influence of non-target substrates (matrix effect) should be considered, as antibodies are sensitive to matrix compounds such as salts, preservatives and solvents. Matrix compounds can lead to unstable antibody assays. It is recommended that researchers test for this early
- If the intended use of the antibody is for immune-based assays, endotoxin levels must be determined. The presence of endotoxins could lead to false positive results, particularly if agonism is being assessed
- Critical results should be confirmed with different, independent antibodies. In this context it is important to realise that often the identical antibody (g. the same clone) is sold by two different manufacturers but without acknowledgement of a common source.
Other helpful information that should be supplied with an antibody include storage conditions and application protocols. A thorough datasheet indicates high quality validation data. Ideally, scientists should always validate commercially-available antibody products in their own laboratory before use. A summary of published antibody validation workflows can be found here.
How to best use antibodies
There are a range of strategies to ensure scientific rigour when using antibodies in early-stage research. These are considered the norm in industry as they help grow confidence in a project and support its progress through the drug development pipeline.
For academic researchers, adopting these practices can galvanise the robustness of results. By operating in line with industry standards, academic researchers can ‘fast track’ their early-stage research towards biopharma investment.
These strategies include:
- Thorough research of commercially-available antibodies by assessing provided validation data and/or reviews on company websites or independent review pages (such as AntibodyPedia) which address the above validation criteria
- Aim to perform an entire series of experiments with antibodies from a single lot. As a biological product, antibodies can vary from one batch to the next
- Use recombinant, monoclonal antibodies where possible
- Use antibodies in an application-specific manner. An antibody that works well for Western blots on denaturing gels, for example, should not be presumed to work equally well in non-denaturing assays such as FACS.
Individually, these strategies are not sufficient to confirm antibody specificity. Rather, multiple tools and directions are key, so these strategies should be employed in concert with one another.
To adequately address this issue of poor quality or misused commercially available antibodies, the entire scientific community, including manufacturers, end users (that is, researchers) and scientific journals, must work together. Only by improving transparency and quality control, will scientists be able to use commercially-available antibodies with confidence, improve the accuracy of their results and accelerate the progress of their project through the development pipeline.
Additional information on issues of antibody reagents is available at: Voskuil JLA, et al. The Antibody Society’s antibody validation webinar series. mAbs 2020; 12: https://doi.org/10.1080/19420862.2020.1794421