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

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...

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Autores principales: Ayoubi, Riham, Ryan, Joel, Biddle, Michael S, Alshafie, Walaa, Fotouhi, Maryam, Bolivar, Sara Gonzalez, Ruiz Moleon, Vera, Eckmann, Peter, Worrall, Donovan, McDowell, Ian, Southern, Kathleen, Reintsch, Wolfgang, Durcan, Thomas M, Brown, Claire, Bandrowski, Anita, Virk, Harvinder, Edwards, Aled M, McPherson, Peter, Laflamme, Carl
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10666931/
https://www.ncbi.nlm.nih.gov/pubmed/37995198
http://dx.doi.org/10.7554/eLife.91645
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author Ayoubi, Riham
Ryan, Joel
Biddle, Michael S
Alshafie, Walaa
Fotouhi, Maryam
Bolivar, Sara Gonzalez
Ruiz Moleon, Vera
Eckmann, Peter
Worrall, Donovan
McDowell, Ian
Southern, Kathleen
Reintsch, Wolfgang
Durcan, Thomas M
Brown, Claire
Bandrowski, Anita
Virk, Harvinder
Edwards, Aled M
McPherson, Peter
Laflamme, Carl
author_facet Ayoubi, Riham
Ryan, Joel
Biddle, Michael S
Alshafie, Walaa
Fotouhi, Maryam
Bolivar, Sara Gonzalez
Ruiz Moleon, Vera
Eckmann, Peter
Worrall, Donovan
McDowell, Ian
Southern, Kathleen
Reintsch, Wolfgang
Durcan, Thomas M
Brown, Claire
Bandrowski, Anita
Virk, Harvinder
Edwards, Aled M
McPherson, Peter
Laflamme, Carl
author_sort Ayoubi, Riham
collection PubMed
description 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.
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spelling pubmed-106669312023-11-23 Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications Ayoubi, Riham Ryan, Joel Biddle, Michael S Alshafie, Walaa Fotouhi, Maryam Bolivar, Sara Gonzalez Ruiz Moleon, Vera Eckmann, Peter Worrall, Donovan McDowell, Ian Southern, Kathleen Reintsch, Wolfgang Durcan, Thomas M Brown, Claire Bandrowski, Anita Virk, Harvinder Edwards, Aled M McPherson, Peter Laflamme, Carl eLife Biochemistry and Chemical Biology 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. eLife Sciences Publications, Ltd 2023-11-23 /pmc/articles/PMC10666931/ /pubmed/37995198 http://dx.doi.org/10.7554/eLife.91645 Text en © 2023, Ayoubi et al https://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Biochemistry and Chemical Biology
Ayoubi, Riham
Ryan, Joel
Biddle, Michael S
Alshafie, Walaa
Fotouhi, Maryam
Bolivar, Sara Gonzalez
Ruiz Moleon, Vera
Eckmann, Peter
Worrall, Donovan
McDowell, Ian
Southern, Kathleen
Reintsch, Wolfgang
Durcan, Thomas M
Brown, Claire
Bandrowski, Anita
Virk, Harvinder
Edwards, Aled M
McPherson, Peter
Laflamme, Carl
Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications
title Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications
title_full Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications
title_fullStr Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications
title_full_unstemmed Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications
title_short Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications
title_sort scaling of an antibody validation procedure enables quantification of antibody performance in major research applications
topic Biochemistry and Chemical Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10666931/
https://www.ncbi.nlm.nih.gov/pubmed/37995198
http://dx.doi.org/10.7554/eLife.91645
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