Systematic Development of Sandwich Immunoassays for the Plasma Secretome.
Proteomics
; 19(15): e1900008, 2019 08.
Article
em En
| MEDLINE
| ID: mdl-31278833
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.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Imunoensaio
Limite:
Humans
/
Middle aged
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article