Antigen retrieval methods for immunohistochemistry have improved and become more standardised over the last 20 years. Detection systems have become more sensitive, streamlined and automated IHC platforms have improved consistency and removed human error. These factors help to maximise the success of immunohistochemical assays. However success remains largely dependent on the availability of reliable, specific antibodies. There is currently no industry standard for the validation of antibodies prior to commercial release, so when a researcher purchases an antibody they must still ask the question is the antibody specific to what it says on the label?
The level of validation across antibody suppliers lies on a continuum and there is no doubt the significant cost implications of validation will be the determining factor.
Usually companies whose central function is to manufacture antibodies have a vested interest in how their products are going to perform, and therefore perform the most comprehensive validations.
Other suppliers use a more “survival of the fittest” approach and sell a wide range of antibodies produced by different manufacturers, offering refunds if the antibody does not work as claimed on the label. Validation is therefore at the expense of the researcher’s time and the quality of that feedback is entirely dependent on the experience of the researcher.
In 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 – the CRO scientists must use their skills and experience to identify the antibodies most likely to be successful, rather than simply believe what it says on the label.
Monya Baker’s 2015 Nature article illustrates that despite the extensive use of antibodies in research, the commercial antibody market is guilty of batch-to-batch variability, unwanted cross-reactivity and poor antibody characterisation. This results in false positive outcomes and reproducibility issues which leads to considerable costs in time, money and often precious samples.
Baker’s report comments that following assessment of the work by the Human Protein Atlas (a Swedish consortium that aims to generate antibodies to every expressed human gene) less than half of the 20,000 commercial antibodies were effective in IHC.
Baker quotes pathologist, David Rimm, a victim of batch-to-batch variability which abruptly scuppered his ground breaking test for skin melanoma, he said that “most scientists believe the label printed on the vial”.
Couchman (2009) conceded a lack of quality control in the production of antibodies and that antibody companies should provide more comprehensive information.
Literature searches can identify antibody clones which have been shown to work well and have been validated by published scientists. Optimal methodologies may also be published. However, often the target antigens are relatively novel and there is little in the literature.
Scrutiny of the supplied datasheets and sourcing a selected range of antibodies is as important in the development of IHC assays as defining the methods in the laboratory. Researchers need to be astute and vigilant when studying the information provided - critically examining any images of labelling and questioning when images are absent. The experience of the researcher is key during the selection of the best antibodies to test.
It is the responsibility of the researcher to have an understanding of scientific / biological functions and localisations of the marker to interpret whether the labelling is specific. It is also their responsibility to include both positive and negative controls and test sufficient variables to ensure that they have given an antibody the opportunity to perform well.