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Proteomics. 2019 Aug;19(15):e1900008. doi: 10.1002/pmic.201900008. Epub 2019 Jul 22.

Systematic Development of Sandwich Immunoassays for the Plasma Secretome.

Author information

1
Division of Affinity Proteomics, Science for Life Laboratory, KTH - Royal Institute of Technology, Box 1031, 171 21, Solna, Sweden.
2
Division of Cellular and Clinical Proteomics, Science for Life Laboratory, KTH - Royal Institute of Technology, Box 1031, 171 21, Solna, Sweden.
3
K.G. Jebsen - Thrombosis Research and Expertise Center (TREC), Department of Clinical Medicine, UiT - The Arctic University of Norway, 9010, Tromsø, Norway.
4
Division of Internal Medicine, University Hospital of North Norway, 9010, Tromsø, Norway.
5
Division of Systems Biology, Science for Life Laboratory, KTH - Royal Institute of Technology, Box 1031, 171 21, Solna, Sweden.
6
Division of Protein Technology, Department of Protein Science, KTH - Royal Institute of Technology, 106 91, Stockholm, Sweden.
7
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2970, Hørsholm, Denmark.
8
Atlas Antibodies AB, 168 69, Bromma, Sweden.

Abstract

The plasma proteome offers a clinically useful window into human health. Recent advances from highly multiplexed assays now call for appropriate pipelines to validate individual candidates. Here, a workflow is developed to build dual binder sandwich immunoassays (SIA) and for proteins predicted to be secreted into plasma. Utilizing suspension bead arrays, ≈1800 unique antibody pairs are first screened against 209 proteins with recombinant proteins as well as EDTA plasma. Employing 624 unique antibodies, dilution-dependent curves in plasma and concentration-dependent curves of full-length proteins for 102 (49%) of the targets are obtained. For 22 protein assays, the longitudinal, interindividual, and technical performance is determined in a set of plasma samples collected from 18 healthy subjects every third month over 1 year. Finally, 14 of these assays are compared with with SIAs composed of other binders, proximity extension assays, and affinity-free targeted mass spectrometry. The workflow provides a multiplexed approach to screen for SIA pairs that suggests using at least three antibodies per target. This design is applicable for a wider range of targets of the plasma proteome, and the assays can be applied for discovery but also to validate emerging candidates derived from other platforms.

KEYWORDS:

antibodies; plasma; sandwich assays; screening; secreted proteins

PMID:
31278833
DOI:
10.1002/pmic.201900008

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