Systematic Development of Sandwich Immunoassays for the Plasma Secretome.

Häussler RS, Bendes A, Iglesias M, 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

Proteomics 19 (15) e1900008 [2019-08-00; online 2019-07-22]

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.

Plasma Profiling [Technology development]

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PubMed 31278833

DOI 10.1002/pmic.201900008

Crossref 10.1002/pmic.201900008