Open Access Methodology

Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification

Jan C Brase1, Heiko Mannsperger1, Holger Fröhlich1, Stephan Gade1, Christian Schmidt1, Stefan Wiemann1, Tim Beissbarth12, Thorsten Schlomm3, Holger Sültmann1 and Ulrike Korf1*

Author Affiliations

1 Division of Molecular Genome Analysis, German Cancer Research Center, Heidelberg, Germany

2 Department of Medical Statistics, University Medicine Göttingen, Göttingen, Germany

3 Martini-Clinic, Prostate Cancer Center, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

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Proteome Science 2010, 8:36  doi:10.1186/1477-5956-8-36

Published: 22 June 2010

Abstract

Background

Reverse phase protein arrays (RPPA) emerged as a useful experimental platform to analyze biological samples in a high-throughput format. Different signal detection methods have been described to generate a quantitative readout on RPPA including the use of fluorescently labeled antibodies. Increasing the sensitivity of RPPA approaches is important since many signaling proteins or posttranslational modifications are present at a low level.

Results

A new antibody-mediated signal amplification (AMSA) strategy relying on sequential incubation steps with fluorescently-labeled secondary antibodies reactive against each other is introduced here. The signal quantification is performed in the near-infrared range. The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89) between AMSA and standard NIR detection. Probing serial dilutions of human cancer cell lines with different primary antibodies demonstrated that the new amplification approach improved the limit of detection especially for low abundant target proteins.

Conclusions

Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs. Contrasting other amplification approaches it allows target protein detection over a large linear range.