==== Front bioRxiv BIORXIV bioRxiv Cold Spring Harbor Laboratory 37398479 10.1101/2023.06.01.543292 preprint 6 Article Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications Ayoubi Riham http://orcid.org/0000-0001-6633-655X Ryan Joel http://orcid.org/0000-0002-7295-815X Biddle Michael S http://orcid.org/0000-0002-9853-8815 Alshafie Walaa http://orcid.org/0000-0001-7999-1297 Fotouhi Maryam http://orcid.org/0000-0001-8937-4719 Bolivar Sara Gonzalez http://orcid.org/0000-0002-4299-8281 Moleon Vera Ruiz http://orcid.org/0000-0003-3728-3158 Eckmann Peter http://orcid.org/0000-0002-5388-9451 Worrall Donovan http://orcid.org/0000-0001-5676-5473 McDowell Ian http://orcid.org/0000-0002-9031-4729 Southern Kathleen http://orcid.org/0000-0002-4125-3608 Reintsch Wolfgang http://orcid.org/0000-0002-5466-7885 Durcan Thomas M http://orcid.org/0000-0003-3942-1956 Brown Claire M http://orcid.org/0000-0003-1622-663X Bandrowski Anita http://orcid.org/0000-0002-5497-0243 Virk Harvinder S http://orcid.org/0000-0002-9739-9593 Edwards Aled M http://orcid.org/0000-0002-4782-6016 McPherson Peter S http://orcid.org/0000-0001-7806-5662 Laflamme Carl http://orcid.org/0000-0001-5906-025X 17 8 2023 2023.06.01.543292http://biorxiv.org/lookup/doi/10.1101/2023.06.01.543292 nihpp-2023.06.01.543292.pdf Antibodies are critical reagents to detect and characterize proteins. It is commonly understood that many commercial antibodies do not recognize their intended targets, but information on the scope of the problem remains largely anecdotal, and as such, feasibility of the goal of at least one potent and specific antibody targeting each protein in a proteome cannot be assessed. Focusing on antibodies for human proteins, we have scaled a standardized characterization approach using parental and knockout cell lines (Laflamme et al., 2019) to assess the performance of 614 commercial antibodies for 65 neuroscience-related proteins. Side-by-side comparisons of all antibodies against each target, obtained from multiple commercial partners, demonstrates that: i) more than 50% of all antibodies failed in one or more tests, ii ) yet, ∼50-75% of the protein set was covered by at least one high-performing antibody, depending on application, suggesting that coverage of human proteins by commercial antibodies is significant; and iii ) recombinant antibodies performed better than monoclonal or polyclonal antibodies. The hundreds of underperforming antibodies identified in this study were found to have been used in a large number of published articles, which should raise alarm. Encouragingly, more than half of the underperforming commercial antibodies were reassessed by the manufacturers, and many had alterations to their recommended usage or were removed from the market. This first such study helps demonstrate the scale of the antibody specificity problem but also suggests an efficient strategy toward achieving coverage of the human proteome; mine the existing commercial antibody repertoire, and use the data to focus new renewable antibody generation efforts. ==== Body pmc