Systematic Development of Sandwich Immunoassays for the Plasma Secretome

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

Keywords: antibodies; plasma; sandwich assays; screening; secreted proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotinylation
  • Humans
  • Immunoassay / methods*
  • Mass Spectrometry
  • Middle Aged
  • Plasma / chemistry
  • Proteome / analysis
  • Proteomics / methods


  • Proteome