Systematic Development of Sandwich Immunoassays for the Plasma Secretome

Research output: Contribution to journalJournal article – Annual report year: 2019Researchpeer-review

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  • Author: Häussler, Ragna S.

    KTH - Royal Institute of Technology, Sweden

  • Author: Bendes, Annika

    KTH - Royal Institute of Technology, Sweden

  • Author: Iglesias, Maria Jesus

    KTH - Royal Institute of Technology, Sweden

  • Author: Sanchez-Rivera, Laura

    KTH - Royal Institute of Technology, Sweden

  • Author: Dodig-Crnković, Tea

    KTH - Royal Institute of Technology, Sweden

  • Author: Byström, Sanna

    KTH - Royal Institute of Technology, Sweden

  • Author: Fredolini, Claudia

    KTH - Royal Institute of Technology, Sweden

  • Author: Birgersson, Elin

    KTH - Royal Institute of Technology, Sweden

  • Author: Dale, Matilda

    KTH - Royal Institute of Technology, Sweden

  • Author: Edfors, Fredrik

    KTH - Royal Institute of Technology, Sweden

  • Author: Fagerberg, Linn

    KTH - Royal Institute of Technology, Sweden

  • Author: Rockberg, Johan

    KTH - Royal Institute of Technology, Sweden

  • Author: Tegel, Hanna

    KTH - Royal Institute of Technology, Sweden

  • Author: Uhlén, Mathias

    High Throughput Molecular Bioscience, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 2800, Kgs. Lyngby, Denmark

  • Author: Qundos, Ulrika

    Atlas Antibodies AB, Sweden

  • Author: Schwenk, Jochen M.

    KTH - Royal Institute of Technology, Sweden

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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.
Original languageEnglish
Article number1900008
JournalProteomics
Volume19
Issue number15
Number of pages19
ISSN1615-9853
DOIs
Publication statusPublished - 2019
CitationsWeb of Science® Times Cited: No match on DOI

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ID: 190216125