1. Systematic Development of Sandwich Immunoassays for the Plasma Secretome.
- Author
-
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, and Schwenk JM
- Subjects
- Biotinylation, Humans, Mass Spectrometry, Middle Aged, Plasma chemistry, Proteome analysis, Proteomics methods, Immunoassay methods
- 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., (© 2019 KTH - Royal Institute of Technology. Proteomics Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2019
- Full Text
- View/download PDF