Antibody Validation… Is There Still A Crisis?
Reliable antibodies are essential: whether you are planning to publish a ground-breaking piece of research, or are carrying out routine assays within your laboratory; reproducibility is key.
Over the years there has been much debate regarding who should take responsibility for antibody validation. Is it the responsibility of the antibody supplier to offer a fully characterised and validated antibody, or should the researcher validate the antibody specifically for their own needs? Initially there was a greater onus on the researcher to check an antibody performed as it should in their assays. Current opinion places a larger obligation on the antibody manufacturer to ensure lot to lot consistency and specificity for the antibody target. As part of our State of the Industry survey published in our 2019 antibody market report, the manufacturers themselves were polled for their opinions on antibody validation. A larger percentage of manufacturers, compared to the previous year, believed antibody validation is a problem that should be tackled by the industry. Only 45.8% of the companies polled in the 2018 survey thought it was an industry problem compared to 57.6% in 2019. When asked to give suggestions on how to improve quality standards, 37% suggested the implementation of global standards.
Leading the way in this endeavour, is an international group of scientists proposing a set of standard validation principles. The International Working Group for Antibody Validation (IWGAV) has suggested 5 ‘pillars’ published by Uhlen, M. et al. in the scientific journal, Nature Methods. These pillars are a set of antibody validation techniques that can be used to ensure antibody reproducibility.
- Genetic validation uses knock-down or knock-out of the target protein as a negative control.
- Orthogonal validation compares protein amounts detected by antibody staining to other protein determination methods.
- Independent antibody validation compares levels of antibody staining verses an independent antibody which is mapped to a different epitope on the same target.
- Recombinant expression validation either contrasts antibody staining of cell lysates from non- and over-expressing cells. Or, compares antibody staining of tagged recombinant target protein to levels detected by an anti-tag antibody.
- Capture MS validation utilises mass spectrometry to analyse proteins captured by the antibody of interest.
A bigger shake up of the research reagent market was presented in the publication by Bradbury and Pluckthun in the scientific journal Nature. Bradbury, Pluckthun and 110 co-signatories called for funding to allow the genetic sequencing of all hybridoma-produced monoclonal antibodies to key targets. Describing inconsistent levels of characterisation between manufacturers, and potential problems associated with hybridomas. They proposed that replacing these antibodies with recombinant versions would ensure a never-ending supply of highly-characterised antibodies. Thus, promoting the transition towards the exclusive use of recombinant antibodies, and even suggesting the phasing out of polyclonal antibodies altogether!
At the 3rd International Antibody Validation Meeting held in Bath, the importance of antigens in antibody generation, characterisation and validation was also stressed. Speakers highlighted the need for them to be full-length, correctly-folded, ‘authentic’ proteins for effective antibody generation. Furthermore, discussions included factors such as antigen abundance and availability, which should be taken into account when planning an antibody characterisation and validation strategy. The role of the scientists purchasing these antibodies was not ignored, with an emphasis on correct usage, as antibody performance is application dependant. The IWGAV approach to antibody validation featured in presentations by some of the biggest players in the research antibody market. These market-leaders were keen to emphasize that they were not only testing their new products to these high validation standards, but were also investigating the quality of their current product catalogues.
This is a great progression for the research antibody market as a whole. Product manufacturers are increasing their breadth and depth of antibody characterisation and validation. Our State of the Industry survey revealed 75.8% of manufacturers have increased the amount of validation on their products over the past year. Companies offering recombinant antibodies are on the increase, and more information that ever is available to scientists. With the development of antibody review platforms such as Antibodypedia, and antibody manufacturers encouraging customers to post product reviews on their websites, scientists will soon have all the tools they need to confidently make informed decisions when purchasing antibodies.
Let us help you – Pivotal Scientific have compiled the most complete list of antibody suppliers that operate in the research market https://pivotalscientific.com/antibody-supplier-list
Uhlen, M. et al. (2016) Nature Methods.13: 823–827
Bradbury, A. and Pluckthun, A. (2015). Nature 518, 27-29
Written by Sharon Craggs – Technical Analyst