%0 Journal Article
%T Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification
%A Jan C Brase
%A Heiko Mannsperger
%A Holger Fr£¿hlich
%A Stephan Gade
%A Christian Schmidt
%A Stefan Wiemann
%A Tim Beissbarth
%A Thorsten Schlomm
%A Holger S¨¹ltmann
%A Ulrike Korf
%J Proteome Science
%D 2010
%I BioMed Central
%R 10.1186/1477-5956-8-36
%X 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.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.In recent years reverse phase protein arrays (RPPA) have proven themselves as useful experimental platform for the validation of biomarker candidate proteins in biological and clinical samples [1-6]. RPPA are considerably faster than conventional techniques such as mass spectrometry, western blotting, 2-D PAGE, and allow the analysis of hundreds of samples in parallel. In addition, measurements can be made with high accuracy and reproducibility. The basic idea of RPPA implies that all samples are spotted in parallel on solid-phase carriers. Samples can be printed either as serial dilutions or in a single concentration but as multiple replicate spots [7]. The detection of a specific protein or a certain phosphorylation site is carried out with a single and highly specific antibody per slide, and the fraction of captured antibodies is mostly visualized with secondary antibodies. Recently, near infrared fluorescence-based detection was reported as useful for reverse phase protein microarrays [8,9]. Routine applications involved analyzing the activation status of signaling pathways
%U http://www.proteomesci.com/content/8/1/36