Advertisement

Highly Multiplexed Antibody Suspension Bead Arrays for Plasma Protein Profiling

  • Kimi Drobin
  • Peter Nilsson
  • Jochen M. Schwenk
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1023)

Abstract

Alongside the increasing availability of affinity reagents, antibody microarrays have become a powerful tool to screen for target proteins in complex samples. Applying directly labeled samples onto arrays instead of using sandwich assays offers an approach to facilitate a systematic, high-throughput, and flexible exploration of protein profiles in body fluids such as serum or plasma. As an alternative to planar arrays, a system based on color-coded beads for the creation of antibody arrays in suspension has become available to offer a microtiter plate-based option for screening larger number of samples with variable sets of capture reagents. A procedure was established for analyzing biotinylated samples without the necessity to remove excess labeling substance. We have shown that this assay system allows detecting proteins down into lower pico-molar and higher pg/ml levels with dynamic ranges over three orders of magnitude. Presently, this workflow enables the profiling of 384 samples for up to 384 proteins per assay.

Key words

Suspension bead array Antibody array Serum Plasma Labeling 

Notes

Acknowledgements

We like to thank the entire staff of the Human Protein Atlas (HPA) for their tremendous efforts within the Human Protein Atlas project. This work is funded by the PRONOVA project (VINNOVA, Swedish Governmental Agency for Innovation Systems), and by grants from the Knut and Alice Wallenberg Foundation and Science for Life Laboratory, Stockholm.

References

  1. 1.
    Hanash S (2003) Disease proteomics. Nature 422:226–232PubMedCrossRefGoogle Scholar
  2. 2.
    Ayoglu B, Haggmark A, Neiman M, Igel U, Uhlen M, Schwenk JM, Nilsson P (2011) Systematic antibody and antigen-based proteomic profiling with microarrays. Expert Rev Mol Diagn 11:219–234PubMedCrossRefGoogle Scholar
  3. 3.
    Kingsmore SF (2006) Multiplexed protein measurement: technologies and applications of protein and antibody arrays. Nat Rev Drug Discov 5:310–320PubMedCrossRefGoogle Scholar
  4. 4.
    Fulton RJ, McDade RL, Smith PL, Kienker LJ, Kettman JR Jr (1997) Advanced multiplexed analysis with the FlowMetrix system. Clin Chem 43:1749–1756PubMedGoogle Scholar
  5. 5.
    Schwenk JM, Gry M, Rimini R, Uhlen M, Nilsson P (2008) Antibody suspension bead arrays within serum proteomics. J Proteome Res 7:3168–3179PubMedCrossRefGoogle Scholar
  6. 6.
    Rimini R, Schwenk JM, Sundberg M, Sjoberg R, Klevebring D, Gry M, Uhlen M, Nilsson P (2009) Validation of serum protein profiles by a dual antibody array approach. J Proteomics 73:252–266PubMedCrossRefGoogle Scholar
  7. 7.
    Schwenk JM, Igel U, Kato BS, Nicholson G, Karpe F, Uhlen M, Nilsson P (2010) Comparative protein profiling of serum and plasma using an antibody suspension bead array approach. Proteomics 10(3):532–540PubMedCrossRefGoogle Scholar
  8. 8.
    Neiman M, Hedberg JJ, Donnes PR, Schuppe-Koistinen I, Hanschke S, Schindler R, Uhlen M, Schwenk JM, Nilsson P (2011) Plasma profiling reveals human fibulin-1 as candidate marker for renal impairment. J Proteome Res 10:4925–4934PubMedCrossRefGoogle Scholar
  9. 9.
    Schwenk JM, Igel U, Kato BS, Nicholson G, Karpe F, Uhlen M, Nilsson P (2010) Comparative protein profiling of serum and plasma using an antibody suspension bead array approach. Proteomics 10:532–540PubMedCrossRefGoogle Scholar
  10. 10.
    Schwenk JM, Igel U, Neiman M, Langen H, Becker C, Bjartell A, Ponten F, Wiklund F, Gronberg H, Nilsson P, Uhlen M (2010) Toward next generation plasma profiling via heat-induced epitope retrieval and array-based assays. Mol Cell Proteomics 9:2497–2507PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Kimi Drobin
    • 1
  • Peter Nilsson
    • 1
  • Jochen M. Schwenk
    • 1
  1. 1.Science for Life Laboratory Stockholm, School of BiotechnologyKTH - Royal Institute of TechnologyStockholmSweden

Personalised recommendations