title: Multiplexed protein profiling by sequential affinity capture.
abstract: Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off-target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi-automated sequential capture assay. This novel bead-based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read-out by a secondary capture bead array. We demonstrate in a proof-of-concept setting that target detection via two sequential affinity interactions reduced off-target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA-based signal amplification, and demonstrate the applicability of the dual capture bead-based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond.
authors.affiliation: Affinity Proteomics, SciLifeLab, School of Biotechnology, KTH - Royal Institute of Technology, Solna, Sweden.
authors.forename: Burcu
authors.initials: B
authors.lastname: Ayoglu
authors.affiliation: Affinity Proteomics, SciLifeLab, School of Biotechnology, KTH - Royal Institute of Technology, Solna, Sweden.
authors.forename: Elin
authors.initials: E
authors.lastname: Birgersson
authors.affiliation: Department of Biochemistry and Biophysics, SciLifeLab, Stockholm University, Solna, Sweden.
authors.forename: Anja
authors.initials: A
authors.lastname: Mezger
authors.affiliation: Department of Biochemistry and Biophysics, SciLifeLab, Stockholm University, Solna, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Nilsson
authors.affiliation: Affinity Proteomics, SciLifeLab, School of Biotechnology, KTH - Royal Institute of Technology, Solna, Sweden.
authors.forename: Mathias
authors.initials: M
authors.lastname: Uhlén
authors.affiliation: Affinity Proteomics, SciLifeLab, School of Biotechnology, KTH - Royal Institute of Technology, Solna, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Nilsson
authors.affiliation: Affinity Proteomics, SciLifeLab, School of Biotechnology, KTH - Royal Institute of Technology, Solna, Sweden.
authors.forename: Jochen M
authors.initials: JM
authors.lastname: Schwenk
mesh_terms: D000941:Antigens / Q000378:metabolism*; D001667:Binding, Competitive; D014408:Biomarkers, Tumor / Q000097:blood / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008280:Magnetics; D008297:Male; D008863:Microspheres; D011471:Prostatic Neoplasms / Q000097:blood / Q000175:diagnosis / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D000941:Antigens; D014408:Biomarkers, Tumor; D020543:Proteome
keywords: Affinity proteomics; Antibody arrays; Plasma profiling; Suspension bead arrays