Abstract

There is a strong need for procedures that enable context and application dependent validation of antibodies. Here, we applied a magnetic bead assisted workflow and immunoprecipitation mass spectrometry (IP-MS/MS) to assess antibody selectivity for the detection of proteins in human plasma. A resource was built on 414 IP experiments using 157 antibodies (targeting 120 unique proteins) in assays with heat-treated or untreated EDTA plasma. For each protein we determined their antibody related degrees of enrichment using z-scores and their frequencies of identification across all IP assays. Out of 1,313 unique endogenous proteins, 426 proteins (33%) were detected in >20% of IPs, and these background components were mainly comprised of proteins from the complement system. For 45% (70/157) of the tested antibodies, the expected target proteins were enriched (z-score ≥ 3). Among these 70 antibodies, 59 (84%) co-enriched other proteins beside the intended target and mainly due to sequence homology or protein abundance. We also detected protein interactions in plasma, and for IGFBP2 confirmed these using several antibodies and sandwich immunoassays. The protein enrichment data with plasma provide a very useful and yet lacking resource for the assessment of antibody selectivity. Our insights will contribute to a more informed use of affinity reagents for plasma proteomics assays.

Details

Title
Systematic assessment of antibody selectivity in plasma based on a resource of enrichment profiles
Author
Fredolini Claudia 1   VIAFID ORCID Logo  ; Byström Sanna 1 ; Sanchez-Rivera, Laura 1 ; Ioannou, Marina 1 ; Tamburro Davide 2 ; Pontén Fredrik 3 ; Branca, Rui M 2 ; Nilsson, Peter 1 ; Lehtiö Janne 2 ; Schwenk, Jochen M 1   VIAFID ORCID Logo 

 KTH - Royal Institute of Technology, Division of Affinity Proteomics, Science for Life Laboratory, Department of Protein Science, Solna, Sweden (GRID:grid.5037.1) (ISNI:0000000121581746) 
 Science for Life Laboratory, Karolinska Institute, Cancer Proteomics, Department of Oncology-Pathology, Solna, Sweden (GRID:grid.4714.6) (ISNI:0000 0004 1937 0626) 
 Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, Department of Immunology, Genetics and Pathology, Uppsala, Sweden (GRID:grid.8993.b) (ISNI:0000 0004 1936 9457) 
Publication year
2019
Publication date
2019
Publisher
Nature Publishing Group
e-ISSN
20452322
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2236156390
Copyright
© The Author(s) 2019. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.