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Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications.

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posted on 2024-01-09, 15:11 authored by Riham Ayoubi, Joel Ryan, Michael S Biddle, Walaa Alshafie, Maryam Fotouhi, Sara Gonzalez Bolivar, Vera Ruiz Moleon, Peter Eckmann, Donovan Worrall, Ian McDowell, Kathleen Southern, Wolfgang Reintsch, Thomas M Durcan, Claire Brown, Anita Bandrowski, Harvinder Virk, Aled M Edwards, Peter McPherson, Carl Laflamme
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, have demonstrated that: (i) more than 50% of all antibodies failed in one or more applications, (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 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.

Funding

Open Drug Discovery Center for Alzheimer's Disease

National Institute on Aging

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Expanding Target Enabling Packages for AMP-AD Target Validation to identify small molecule inhibitors that block protein-protein interactions in cells

National Institute on Aging

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FDN154305

OGI-210

EUbOPEN: Enabling and Unlocking biology in the OPEN

European Commission

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History

Citation

Riham AyoubiJoel RyanMichael S BiddleWalaa AlshafieMaryam FotouhiSara Gonzalez BolivarVera Ruiz MoleonPeter EckmannDonovan WorrallIan McDowellKathleen SouthernWolfgang ReintschThomas M DurcanClaire BrownAnita BandrowskiHarvinder VirkAled M EdwardsPeter McPhersonCarl Laflamme (2023) Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications eLife 12:RP91645. https://doi.org/10.7554/eLife.91645.2

Author affiliation

Department of Respiratory Sciences, University of Leicester

Version

  • VoR (Version of Record)

Published in

eLife

Volume

12

Pagination

RP91645

Publisher

eLife Sciences Publications, Ltd

issn

2050-084X

eissn

2050-084X

Acceptance date

2023-10-05

Copyright date

2023

Available date

2024-01-09

Spatial coverage

England

Language

eng

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