Abstract
In recent years, antibody microarrays have developed into an important tool for proteomics. As a multiplexing technique, they facilitate the highly parallel detection of hundreds of different analytes from very small sample volumes of only a few microlitres. This is combined with high sensitivity in the picomolar to femtomolar range, which is similar to the sensitivity of ELISA, the gold standard for protein quantification. In order to obtain such sensitivities in a robust and reproducible manner for sets of several hundreds of analytes simultaneously, it is essential to optimise the experimental layout, sample handling, labelling and incubation as well as data processing steps. Here, we present our current antibody microarray protocols for multiplexed expression profiling studies, which permit the analysis of the abundance of more than 800 proteins in plasma, urine and tissue samples.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Dudley AM, Aach J, Steffen MA, Church GM (2002) Measuring absolute expression with microarrays with a calibrated reference sample and an extended signal intensity range. Proc Natl Acad Sci USA 99:7554–7559
Fellenberg K, Busold CH, Witt O, Bauer A, Beckmann B, Hauser NC, Frohme M, Winter S, Dippon J, Hoheisel JD (2006) Systematic interpretation of microarray data using experiment annotations. BMC Genomics 7:319
Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Tacus S, Trizary R, Leisch F, Li C, Maeschler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JYH, Zhang J (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5:R80
Kapushesky M, Kemmeren P, Culhane AC, Durinck S, Ihmels J, Körner C, Kull M, Torrente A, Sarkans U, Vilo J et al (2004) Expression Profiler: next generation-an online platform for analysis of microarray data. Nucleic Acids Res 32:W465–W470
Kusnezow W, Jacob A, Walijew A, Diehl F, Hoheisel JD (2003) Antibody microarrays: an evaluation of production parameters. Proteomics 3:254–264
Kusnezow W, Syagailo YV, Rüffer S, Baudenstiel N, Gauer C, Hoheisel JD, Wild D, Goychuk I (2006a) Optimal design of microarray immunoassays to compensate for kinetic limitations: theory and experiment. Mol Cell Proteomics 5:1681–1696
Kusnezow W, Syagailo YV, Rüffer S, Klenin K, Sebald W, Hoheisel JD, Gauer C, Goychuk I (2006b) Kinetics of antigen binding to antibody microspots: strong limitation by mass transport to the surface. Proteomics 6:794–803
Kusnezow W, Banzon V, Schröder C, Schaal R, Hoheisel JD, Rüffer S, Luft P, Duschl A, Syagailo YV (2007) Antibody microarray-based profiling of complex specimens: systematic evaluation of labelling strategies. Proteomics 7:1786–1799
Saeed AI, Sharov V, White J, Li J, Liang W, Bhagabati N, Braisted J, Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Trush V, Quackenbush J (2003) TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34:374–378
Schweitzer B, Wiltshire S, Lambert J, O'Malley S, Kukan-skis K, Zhu Z, Kingsmore SF, Lizardi PM, Ward DC (2000) Inaugural article: immunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection. Proc Natl Acad Sci USA 97:10113–10119
Wingren C, Ingvarsson J, Dexlin L, Szul D, Borrebaeck CAK (2007) Design of recombinant antibody microarrays for complex proteome analysis: choice of sample labelling-tag and solid support. Proteomics 7:3055–3065
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer-Verlag Berlin Heidelberg
About this protocol
Cite this protocol
Schröder, C., Jacob, A., Rüffer, S., Fellenberg, K., Hoheisel, J.D. (2010). Antibody Microarrays for Expression Analysis. In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-01147-4_33
Download citation
DOI: https://doi.org/10.1007/978-3-642-01147-4_33
Publisher Name: Springer, Berlin, Heidelberg
Print ISBN: 978-3-642-01146-7
Online ISBN: 978-3-642-01147-4
eBook Packages: Springer Protocols