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Sci Rep. 2019 Jun 6;9(1):8324. doi: 10.1038/s41598-019-43552-5.

Systematic assessment of antibody selectivity in plasma based on a resource of enrichment profiles.

Author information

1
Division of Affinity Proteomics, Science for Life Laboratory, Department of Protein Science, KTH - Royal Institute of Technology, 171 21, Solna, Sweden.
2
Cancer Proteomics, Department of Oncology-Pathology, Science for Life Laboratory, Karolinska Institute, 171 21, Solna, Sweden.
3
Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Rudbeck Laboratory, Uppsala University, 751 85, Uppsala, Sweden.
4
Division of Affinity Proteomics, Science for Life Laboratory, Department of Protein Science, KTH - Royal Institute of Technology, 171 21, Solna, Sweden. jochen.schwenk@scilifelab.se.

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.

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