Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

TitleAssessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.
Publication TypeJournal Article
Year of Publication2015
AuthorsMarcon, E., H. Jain, A. Bhattacharya, H. Guo, S. Phanse, S. Pu, G. Byram, B. C. Collins, E. Dowdell, M. Fenner, X. Guo, A. Hutchinson, J. J. Kennedy, B. Krastins, B. Larsen, Z-Y. Lin, M. F. Lopez, P. Loppnau, S. Miersch, T. Nguyen, J. B. Olsen, M. Paduch, M. Ravichandran, A. Seitova, G. Vadali, M. S. Vogelsang, J. R. Whiteaker, G. Zhong, N. Zhong, L. Zhao, R. Aebersold, C. H. Arrowsmith, A. Emili, L. Frappier, A-C. Gingras, M. Gstaiger, A. G. Paulovich, S. Koide, A. A. Kossiakoff, S. S. Sidhu, S. J. Wodak, S. Gräslund, J. F. Greenblatt, and A. M. Edwards
JournalNat Methods
Volume12
Issue8
Pagination725-31
Date Published2015 Aug
ISSN1548-7105
KeywordsAntibodies, Monoclonal, Antibody Specificity, Chromatin, Cloning, Molecular, Computational Biology, Escherichia coli, HEK293 Cells, Humans, Immunoglobulin Fragments, Immunoglobulin G, Immunoprecipitation, Mass Spectrometry, Peptide Library, Proteins, Proteome, Proteomics, Reproducibility of Results
Abstract

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as 'IP gold standard'. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.

DOI10.1038/nmeth.3472
Alternate JournalNat. Methods
PubMed ID26121405
Research group: