For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome

Hello. We're improving abcam.com and we'd welcome your feedback.

Hello. We're improving abcam.com and we'd welcome your feedback.

Infomation icon

We haven't added this to the BETA yet

New BETA website

New BETA website

Hello. We're improving abcam.com and we'd welcome your feedback.

Take a look at our BETA site and see what we’ve done so far.

Switch on our new BETA site

Now available

Search and browse selected products

  • A selection of primary antibodies

Purchase these through your usual distributor

In the coming months

  • Additional product types
  • Supporting content
  • Sign in to your account
  • Purchase online
United States
Your country/region is currently set to:

If incorrect, please enter your country/region into the box below, to view site information related to your country/region.

Call (888) 77-ABCAM (22226) or contact us
Need help? Contact us

  • My account
  • Sign out
Sign in or Register with us

Welcome

Sign in or

Don't have an account?

Register with us
My basket
Quick order
Abcam homepage

  • Research Products
    By product type
    Primary antibodies
    Secondary antibodies
    ELISA and Matched Antibody Pair Kits
    Cell and tissue imaging tools
    Cellular and biochemical assays
    Proteins and Peptides
    By product type
    Proteomics tools
    Agonists, activators, antagonists and inhibitors
    Cell lines and Lysates
    Multiplex miRNA assays
    Multiplex Assays
    By research area
    Cancer
    Cardiovascular
    Cell Biology
    Epigenetics
    Metabolism
    Developmental Biology
    By research area
    Immunology
    Microbiology
    Neuroscience
    Signal Transduction
    Stem Cells
  • Customized Products & Partnerships
    Customized Products & Partnerships

    Customized products and commercial partnerships to accelerate your diagnostic and therapeutic programs.

    Customized products

    Partner with us

  • Support
    Support hub

    Access advice and support for any research roadblock

    View support hub

    Protocols

    Your experiments laid out step by step

    View protocols

  • Events
    • Conference calendar
    • Cancer
    • Cardiovascular
    • Epigenetics & Nuclear signaling
    • Immunology
    • Neuroscience
    • Stem cells
    • Tradeshows
    • Scientific webinars
    Keep up to date with the latest events

    Full event breakdown with abstracts, speakers, registration and more

    View global event calendar

  • Pathways
    Cell signalling pathways

    View all pathways

    View all interactive pathways

Knock-out validation: Overcoming the perils of poor antibody specificity

Related

  • KO validation for antibody specificity
    • Webinar: Raising standards with KO technology
      • Webinar: Validating antibodies with KO
        • Knock-out cell lines
          • Fast-track your research with pre-made KO cell lines
            • Applying CRISPR to the drug discovery pipeline
              • Accelerate the drug pipeline with CRISPR
                • CRISPR technology to streamline assay development
                  • Custom cell engineering services
                    • Custom cell lines: Factors to consider before you begin
                      • Knock-out cell lysates
                        • KO cell line or lysate? Which do I need?

                          We discuss how poor antibody validation has impacted research and development across the life sciences and how the use of knock-outs can help you get this right first time in your work.

                          Over the past decade, the number of studies highlighting poor antibody validation as an underlying issue of research reproducibility in life science has increased 1-4. Our antibodies are extensively validated across many applications, but antibodies must also be validated by the end-user in their own experimental set-up to ensure functionality.

                          As research is frequently built on previous studies, unreliable data from one study may be used to support further research and development. This house of cards, based on non-specific and poorly validated antibodies, has tumbled in many cases, often at the expense of years of research. Over 36% of unproductive experiments in preclinical R&D can be attributed to ineffective reagents, including antibodies. This reagent-related irreproducibility is costing the life science industry over $17 billion annually 2

                            How does poor antibody validation affect published research?

                            There are numerous examples of studies failing due to poorly validated antibodies. In some cases, the failure of the antibody becomes apparent during the project and researchers must go back to the drawing board. For others, this has meant retracting published studies from journals, with no way to assess the wider downstream impact on research that has been based on such published studies.

                            In 2006, a paper published in EMBO Journal, identified the post-translational regulatory mechanisms of the transcriptional repressor methyl-CpG-binding domain protein 1 (MBD1) 5. Since its publication, this paper was cited by 30 other articles. However, in 2019, the paper was retracted due to the demonstrated off-target specificity of the antibody used in this study 6. The article retraction shows that rather than binding to the supposed target of MBD1, the antibody used in the study actually bound specifically to a different protein, cleavage, and polyadenylation specificity factor 6 (CPSF6) 3.

                            A more recent study from Novartis, published in Nature Cell Biology, was cited in 31 other studies and then retracted three years after publication, due to the lack of specificity of a number of antibodies used in the study, which called into question the paper’s main conclusions.7

                            These are not isolated cases. In a field-wide call for increased antibody standards, Bradbury and Plückthun stated that poorly characterized antibodies were largely responsible for the failure to replicate more than 11% of landmark preclinical studies 3,4. A study from the University of Toronto, Canada, assessing 1124 affinity reagents targeting chromatin-related proteins, revealed that just 31% were specific for their target antigen 8. Indeed, a recent analysis suggests that the high rate of attrition in drug development is due to poorly validated research reagents, including antibodies, in the preclinical phase 2.

                            The problem has become so widespread that journals are now publishing guidelines for researchers requiring antibody validation data before publishing antibody-based studies 9. However, while antibody identification by researchers has improved in response to journal specifications, researchers are still failing to perform adequate experimental validation 10,11.

                            How do knock-outs improve antibody validation?

                            The industry gold standard for ensuring antibody specificity and overcoming these issues is knock-out (KO) validation. Using a KO cell line that does not express your protein of interest offers a true negative control. While many suppliers validate their antibodies extensively for use in different applications, each experimental set-up is different and requires end-user validation to confirm the antibody’s performance 1, 12-14.

                            At Abcam, we use large-scale KO validation to confirm the specificity of our antibodies. From this, we have learned that validating antibodies in multiple applications – from western blot to immunocytochemistry – is essential to ensure the specificity of an antibody for each intended method. Antibody specificity is highly dependent upon the assay the antibody is used in; assay conditions may affect antibody binding, and the presentation of the antigen, whether in folded or denatured form, can determine whether an antibody will detect the protein target 1, 12-14.

                            A study from a team at the Montreal Neurological Institute and Hospital, Canada, showed how the use of CRISPR KO cells could help to select antibodies that are target specific and function in relevant applications. The researchers generated a CRISPR-Cas9 KO cell line for their target, the major amyotrophic lateral sclerosis (ALS) disease gene C9ORF72. They then used this to test a panel of 16 commercially available antibodies to C9ORF72 via several techniques. Out of the 16 different antibodies, only one was specific for the target in immunofluorescence, while a further two showed target specificity via other techniques 15.

                            Another key lesson reflected in the research is that a well-cited antibody does not necessarily translate to a well-validated antibody. The study from Montreal on C9ORF72 showed that the antibodies identified as target specific had not been cited in the research. In contrast, those that were non-specific were widely cited for the target. In-house we have seen similar results; for example, a popular CD73 antibody showed off-target interactions, labeling a protein at 55 kDa rather than the 69-73 kDa expected for CD73, while the KO-validated antibody that demonstrated target specificity was not as highly cited. This resulted in the removal of the non-specific antibody from our catalog, to ensure the accuracy of our reagents from researchers.

                            The call for the use of KO cell lines to validate antibody specificity and help overcome the problems of research retraction, failure of preclinical trials to validate targets and the time and cost of wasted research is increasing 1, 8-10. As journals request thorough end-user validation of antibodies for publication 8, and both academic and industrial researchers alike hope to improve validation standards to increase the translation of their research, KO cell lines and KO lysates are being recognized as the right solution to the problem of antibody specificity.


                            In addition to in-house knock-out validation of our antibodies, we have developed a range of KO cell lines and lysates to support antibody validation in your own experimental system. All our KO cell lines are Sanger sequenced to confirm ablation, with many further validated at the proteomic level via western blot. For rapid analysis, we offer KO cell lysates, so that you can get straight to validating without the need for cell culture, or for more intensive methods you can purchase a KO cell line. We can supply a WT sample for all our KO cell lines and lysates to make sure you have everything you need to fully validate your antibody and ensure the reliability of your data.

                            Browse the KO catalog


                            References

                            1. Bordeaux, J. et al. Biotechniques 48(3):197-209 (2010)
                            2. BenchSci. [Accessed 15/06/2020] (2020)
                            3. Bradbury, A. & Plückthun, A. Nature 518(7537):27-9 (2015)
                            4. Begley, C. G. & Ellis, L. M. Nature 483, 531–533 (2012)
                            5. Lyst, M.J. et al. EMBO J 25(22): 5317–5328 (2006)
                            6. Lyst et al. EMBO J. 38(20): e103220 (2019)
                            7. Fonseca, S.G. et al. Nat Cell Biol 17(1):105 (2015)
                            8. Marcon, E. et al. Nature 12(8):725-31 (2015)
                            9. Brooke, H.L. & Lindsey, M.L. Am J Physiol Heart Circ Physiol 314(4):H724-H732 (2018)
                            10. Gautron, L. Front Neuroanat 13: 46. (2019)
                            11. Hoek, J.M. et al. PeerJ 8:e9300 (2020)
                            12. Dove, A. Science 357:1165-7 (2017)
                            13. Sfanos, K.S. et al. Asian J Urol 6(1): 10–25 (2019)
                            14. Berglund, L. et al. Mol Cell Proteomics 7:2019-27 (2008)
                            15. Laflamme, C. et al. eLife 8:e48363 (2019)





                            Get resources and offers direct to your inbox Sign up
                            A-Z by research area
                            • Cancer
                            • Cardiovascular
                            • Cell biology
                            • Developmental biology
                            • Epigenetics & Nuclear signaling
                            • Immunology
                            • Metabolism
                            • Microbiology
                            • Neuroscience
                            • Signal transduction
                            • Stem cells
                            A-Z by product type
                            • Primary antibodies
                            • Secondary antibodies
                            • Biochemicals
                            • Isotype controls
                            • Flow cytometry multi-color selector
                            • Kits
                            • Loading controls
                            • Lysates
                            • Peptides
                            • Proteins
                            • Slides
                            • Tags and cell markers
                            • Tools & Reagents
                            Help & support
                            • Support
                            • Make an Inquiry
                            • Protocols & troubleshooting
                            • Placing an order
                            • RabMAb products
                            • Biochemical product FAQs
                            • Training
                            • Browse by Target
                            Company
                            • Corporate site
                            • Investor relations
                            • Company news
                            • Careers
                            • About us
                            • Blog
                            Events
                            • Tradeshows
                            • Conferences
                            International websites
                            • abcam.cn
                            • abcam.co.jp

                            Join with us

                            • LinkedIn
                            • facebook
                            • Twitter
                            • YouTube
                            • Terms of sale
                            • Website terms of use
                            • Cookie policy
                            • Privacy policy
                            • Legal
                            • Modern slavery statement
                            © 1998-2023 Abcam plc. All rights reserved.