Bead-Based and Multiplexed Immunoassays for Protein Profiling via Sequential Affinity Capture

  • Elin Birgersson
  • Jochen M. SchwenkEmail author
  • Burcu Ayoglu
Part of the Methods in Molecular Biology book series (MIMB, volume 1619)


Antibody microarrays offer high-throughput immunoassays for multiplexed analyses of clinical samples. For such approaches, samples are either labeled in solution to enable a direct readout on the single binder assay format or detected by matched pairs of capture and detection antibodies in dual binder assay format, also known as sandwich assays. Aiming to benefit from the flexibility and capacity offered by single binder assay readout and the specificity and sensitivity of dual binder assays, we developed a multiplexed dual binder procedure that is based on a sequential, rather than combined, antigen binding. The method, entitled dual capture assay (DCA), is composed of an initial antigen capture by antibodies on beads, followed by labeling of captured protein targets on beads, combinatorial elution steps at high and low pH, and a readout using a secondary bead array. Compared to classical single binder assays, the described method demonstrated several advantages such as reduced contribution of off-target binding, lower noise levels, and improved correlation when comparing with clinical reference values. This procedure describes a novel and versatile immunoassay strategy for proteome profiling in body fluids.

Key words

Affinity proteomics Antibody arrays Plasma profiling Suspension bead array Antibody selectivity Assay sensitivity 



We thank all members of the Biobank Profiling group at SciLifeLab, as well as the entire staff of the Human Protein Atlas. This study was funded by grants from Science for Life Laboratory, the Knut and Alice Wallenberg Foundation, and the KTH Center for Applied Proteomics (KCAP) funded by the Erling-Persson Family Foundation. The authors declare no conflict of interest.


  1. 1.
    Solier C, Langen H (2014) Antibody-based proteomics and biomarker research-current status and limitations. Proteomics 14:774–783CrossRefPubMedGoogle Scholar
  2. 2.
    Fu Q, Schoenhoff FS, Savage WJ, Zhang P, Van Eyk JE (2010) Multiplex assays for biomarker research and clinical application: translational science coming of age. Proteomics Clin Appl 4:271–284CrossRefPubMedGoogle Scholar
  3. 3.
    Uhlen M, Fagerberg L, Hallstrom BM et al (2015) Tissue-based map of the human proteome. Science 347:1260419–1260419CrossRefPubMedGoogle Scholar
  4. 4.
    Ayoglu B, Haggmark A, Neiman M et al (2011) Systematic antibody and antigen-based proteomic profiling with microarrays. Expert Rev Mol Diagn 11:219–234CrossRefPubMedGoogle Scholar
  5. 5.
    Bystrom S, Ayoglu B, Haggmark A et al (2014) Affinity proteomic profiling of plasma, cerebrospinal fluid, and brain tissue within multiple sclerosis. J Proteome Res 13:4607–4619CrossRefPubMedGoogle Scholar
  6. 6.
    Ayoglu B, Chaouch A, Lochmüller H et al (2014) Affinity proteomics within rare diseases: a BIO-NMD study for blood biomarkers of muscular dystrophies. EMBO Mol Med 6:918–936CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Bachmann J, Burté F, Pramana S et al (2014) Affinity proteomics reveals elevated muscle proteins in plasma of children with cerebral malaria. PLoS Pathog 10:e1004038CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Landegren U, Vänelid J, Hammond M et al (2012) Opportunities for sensitive plasma proteome analysis. Anal Chem 84:1824–1830CrossRefPubMedGoogle Scholar
  9. 9.
    Ayoglu B, Birgersson E, Mezger A et al (2016) Multiplexed protein profiling by sequential affinity capture. Proteomics 16:1251–1256CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Zhou S, Lu X, Chen C, Sun D (2010) An immunoassay method for quantitative detection of proteins using single antibodies. Anal Biochem 400:213–218CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Elin Birgersson
    • 1
  • Jochen M. Schwenk
    • 1
    Email author
  • Burcu Ayoglu
    • 1
  1. 1.Affinity Proteomics, SciLifeLab, School of BiotechnologyKTH – Royal Institute of TechnologySolnaSweden

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