Systematic Development of Sandwich Immunoassays for the Plasma Secretome

Ragna S. Häussler, Annika Bendes, Maria Jesus Iglesias, Laura Sanchez-Rivera, Tea Dodig-Crnković, Sanna Byström, Claudia Fredolini, Elin Birgersson, Matilda Dale, Fredrik Edfors, Linn Fagerberg, Johan Rockberg, Hanna Tegel, Mathias Uhlén, Ulrika Qundos, Jochen M. Schwenk

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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.
Original languageEnglish
Article number1900008
JournalProteomics
Volume19
Issue number15
Number of pages19
ISSN1615-9853
DOIs
Publication statusPublished - 2019

Cite this

Häussler, R. S., Bendes, A., Iglesias, M. J., Sanchez-Rivera, L., Dodig-Crnković, T., Byström, S., ... Schwenk, J. M. (2019). Systematic Development of Sandwich Immunoassays for the Plasma Secretome. Proteomics, 19(15), [1900008]. https://doi.org/10.1002/pmic.201900008
Häussler, Ragna S. ; Bendes, Annika ; Iglesias, Maria Jesus ; Sanchez-Rivera, Laura ; Dodig-Crnković, Tea ; Byström, Sanna ; Fredolini, Claudia ; Birgersson, Elin ; Dale, Matilda ; Edfors, Fredrik ; Fagerberg, Linn ; Rockberg, Johan ; Tegel, Hanna ; Uhlén, Mathias ; Qundos, Ulrika ; Schwenk, Jochen M. / Systematic Development of Sandwich Immunoassays for the Plasma Secretome. In: Proteomics. 2019 ; Vol. 19, No. 15.
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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.",
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Häussler, RS, Bendes, A, Iglesias, MJ, Sanchez-Rivera, L, Dodig-Crnković, T, Byström, S, Fredolini, C, Birgersson, E, Dale, M, Edfors, F, Fagerberg, L, Rockberg, J, Tegel, H, Uhlén, M, Qundos, U & Schwenk, JM 2019, 'Systematic Development of Sandwich Immunoassays for the Plasma Secretome', Proteomics, vol. 19, no. 15, 1900008. https://doi.org/10.1002/pmic.201900008

Systematic Development of Sandwich Immunoassays for the Plasma Secretome. / Häussler, Ragna S.; Bendes, Annika; Iglesias, Maria Jesus; Sanchez-Rivera, Laura; Dodig-Crnković, Tea; Byström, Sanna; Fredolini, Claudia; Birgersson, Elin; Dale, Matilda; Edfors, Fredrik; Fagerberg, Linn; Rockberg, Johan; Tegel, Hanna; Uhlén, Mathias; Qundos, Ulrika; Schwenk, Jochen M.

In: Proteomics, Vol. 19, No. 15, 1900008, 2019.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Systematic Development of Sandwich Immunoassays for the Plasma Secretome

AU - Häussler, Ragna S.

AU - Bendes, Annika

AU - Iglesias, Maria Jesus

AU - Sanchez-Rivera, Laura

AU - Dodig-Crnković, Tea

AU - Byström, Sanna

AU - Fredolini, Claudia

AU - Birgersson, Elin

AU - Dale, Matilda

AU - Edfors, Fredrik

AU - Fagerberg, Linn

AU - Rockberg, Johan

AU - Tegel, Hanna

AU - Uhlén, Mathias

AU - Qundos, Ulrika

AU - Schwenk, Jochen M.

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

U2 - 10.1002/pmic.201900008

DO - 10.1002/pmic.201900008

M3 - Journal article

VL - 19

JO - Proteomics

JF - Proteomics

SN - 1615-9853

IS - 15

M1 - 1900008

ER -

Häussler RS, Bendes A, Iglesias MJ, Sanchez-Rivera L, Dodig-Crnković T, Byström S et al. Systematic Development of Sandwich Immunoassays for the Plasma Secretome. Proteomics. 2019;19(15). 1900008. https://doi.org/10.1002/pmic.201900008