Antibody Validation: Does It Do What It Says on the Label?
Enhancing Research Reproducibility, Minimizing Costs, and Selecting Reliable Antibodies
The IHC Standard
Over the last 20 years, antigen retrieval methods for immunohistochemistry (IHC) have improved and become increasingly standardised. Similarly, detection systems have become more sensitive and streamlined whilst the development of automated IHC platforms has improved run consistency and has largely removed human error. These factors have helped to maximise the success of immunohistochemical assays; however, IHC success remains largely dependent on the availability of reliable, target specific, antibodies. There is currently no industry standard for the validation of antibodies prior to commercial release.
As such, when a researcher purchases an antibody, they must still ask the question – is the antibody specific to what it says on the label?
The extent of validation amongst antibody suppliers varies along a continuum, with the decisive factor often being the associated cost implications. Some suppliers, particularly those whose primary focus is antibody production, prioritise and report rigorous validation processes before introducing their products to the scientific research community. They have a vested interest in ensuring their antibodies perform reliably in the field. In contrast, other suppliers adopt a more “survival of the fittest” approach, offering a wide array of antibodies from different manufacturers and providing guarantees and refunds if the antibody fails to meet its label claims. In such cases, validation becomes the responsibility of the researcher, consuming their time and hinging on their expertise for quality feedback.
According to Baker (2015), it is demonstrated that despite the prominent use of antibodies in research, the commercial antibody market is guilty of batch-to-batch variability, unwanted cross-reactivity, and poor antibody characterisation. This leads to false positive results and reproducibility problems throughout the field, incurring significant time, financial, and sample-related costs.
Taussig (2018) reported that many research antibodies fail to live up to expectations, e.g. by not recognising the protein they are supposed to, by recognising a different protein instead of, or in addition to, the desired target, or functioning in some applications but not others.
Contract research timelines and budgets are tight and selecting a validated antibody can make the difference between a project’s success or failure. It is often beyond the scope of contract studies to test multiple antibodies against the marker of interest to find an antibody that does what it says on the label. It may therefore prove to be a false economy to initially choose a cheaper antibody with no reported validation information as failed assays may require repeating or a replacement antibody ultimately needing to be sourced.
Both Baker (2015) and Taussig (2018) highlight that certain scientists contend that up to half of commercially available antibodies lack reliability, based on an evaluation conducted by the Human Protein Atlas—a Swedish consortium with the goal of producing antibodies for every human genome gene. Their assessment revealed that only approximately half of the 55,000 commercial polyclonal antibodies and 5000 monoclonal antibodies tested proved effective in immunohistochemistry and immunocytochemistry. Couchman (2009) suggests that the lack of quality control in antibody production contributes to these problems, underscoring the importance of antibody companies offering more comprehensive information.
Literature searches can reveal key reagents and well-known antibody clones which have been shown to work well and ‘validated’ by published scientists. However, in contract research, the markers of interest are often novel and therefore datasheet scrutiny is the only option.
As a result, sourcing antibodies and selecting the best candidates to test is as important in the development of IHC assays as defining the optimised method within the laboratory. The overarching objective of antibody-based research and diagnostics is to be able to select ‘the correct reagent for a particular target for a specific application’ (Taussig, 2018).
Extra Human Intervention
Baker (2015) quotes pathologist, David Rimm, a victim of batch-to-batch variability which abruptly scuppered his ground-breaking test for skin melanoma, “most scientists believe the label printed on the vial”.
Researchers must remain vigilant and discerning when reviewing information from antibody suppliers. It’s important to carefully assess labelling images and question situations where images are missing. Experience (and intuition gained from experience) are crucial in choosing the right candidates for testing.
It is the responsibility of the researcher to understand scientific / biological functions and localisations of the marker to interpret whether the labelling is specific. Antibody characterisation and validation can be regarded as parallel requirements which go hand in hand in determining the properties governing the use of antibodies in different applications (Taussig, 2018). It is also the responsibility of the researcher to include both suitably fixed, good quality positive and negative controls and to test sufficient variables to ensure they have given an antibody the opportunity to perform well.
What Epistem Offers
Epistem has a highly experienced histology team with over 15 years of expertise in antibody validation and optimisation. Our histology services can be offered independently or integrated as needed, all conducted within a GCLP-Accredited and HTA Registered Laboratory.
Offering a versatile approach to marker selection, Epistem accommodates both well-established and less familiar antigens. We have the capability to either procure antibodies or collaborate with clients using their own antibodies. Furthermore, we can source appropriate control tissues from our collection, comprising normal and diseased tissues, tumour xenografts, cell pellets, and tissues from other species.
Leveraging a deep understanding of negative and positive controls, antigen retrieval techniques, and detection methods, Epistem have the capability to design protocols that can be fine-tuned or validated to meet specific requirements. These protocols can be executed manually on the benchtop or on an automated open system or via the Ventana Discovery Ultra IHC automated platform, ensuring enhanced standardisation. With a continually expanding portfolio of over 150 antibodies, along with fully optimised protocols for human and rodent tissues, Epistem is well-equipped to address diverse histology needs. Additionally, we offer high-resolution digital images of H&E / Tinctorial special stains and IHC labelled slides, facilitating remote viewing and sharing on your preferred platform.
All IHC images can be further interrogated to enable quantification of the extent and intensity of labelling using the Aperio® ScanScope®. Image scanning, analytical quantification and archiving are all fully GCLP compliant.
If you have an antibody and marker of interest, please get in touch and we would be happy to discuss this with you.
Taussig, M (2018). Antibody Validation: a view from the mountains. N Biotechnol. October 25; 45: 1-8. doi: https://doi.org/10.1016/j.nbt.2018.08.002
Baker, M. (2015). Reproducibility crisis: Blame it on the antibodies. Nature, 521(7552), pp.274–276. doi: https://doi.org/10.1038/521274a
Couchman, J.R. (2008). Commercial Antibodies: The Good, Bad, and Really Ugly. Journal of Histochemistry & Cytochemistry, 57(1), pp.7–8. doi: https://doi.org/10.1369/jhc.2008.952820