title: Selectivity analysis of single binder assays used in plasma protein profiling.
abstract: The increasing availability of antibodies toward human proteins enables broad explorations of the proteomic landscape in cells, tissues, and body fluids. This includes assays with antibody suspension bead arrays that generate protein profiles of plasma samples by flow cytometer analysis. However, antibody selectivity is context dependent so it is necessary to corroborate on-target detection over off-target binding. To address this, we describe a concept to directly verify interactions from antibody-coupled beads by analysis of their eluates by Western blots and MS. We demonstrate selective antibody binding in complex samples with antibodies toward a set of chosen proteins with different abundance in plasma and serum, and illustrate the need to adjust sample and bead concentrations accordingly. The presented approach will serve as an important tool for resolving differential protein profiles from antibody arrays within plasma biomarker discoveries.
authors.affiliation: Science for Life Laboratory, School of Biotechnology, KTH-Royal Institute of Technology, Solna, Sweden.
authors.forename: Maja
authors.initials: M
authors.lastname: Neiman
authors.forename: Claudia
authors.initials: C
authors.lastname: Fredolini
authors.forename: Henrik
authors.initials: H
authors.lastname: Johansson
authors.forename: Janne
authors.initials: J
authors.lastname: Lehtiö
authors.forename: Per-Åke
authors.initials: PÅ
authors.lastname: Nygren
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.forename: Peter
authors.initials: P
authors.lastname: Nilsson
authors.forename: Jochen M
authors.initials: JM
authors.lastname: Schwenk
mesh_terms: D000209:Acute-Phase Proteins; D055910:Antibodies, Immobilized / Q000737:chemistry*; D000918:Antibody Specificity; D001798:Blood Proteins / Q000378:metabolism*; D019742:Chemokines, CC / Q000097:blood; D030562:Databases, Protein; D006801:Humans; D000071068:Lipocalin-2; D054834:Lipocalins / Q000097:blood; D040901:Proteomics; D011518:Proto-Oncogene Proteins / Q000097:blood
chemical_list: D000209:Acute-Phase Proteins; D055910:Antibodies, Immobilized; D001798:Blood Proteins; C481939:CCL16 protein, human; D019742:Chemokines, CC; C489506:LCN2 protein, human; D000071068:Lipocalin-2; D054834:Lipocalins; D011518:Proto-Oncogene Proteins
keywords: Antibodies; Plasma profiling; Protein arrays; Suspension bead arrays; Verification