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

Brase JC, Mannsperger H, Fröhlich H, Gade S, Schmidt C, Wiemann S, Beissbarth T, Schlomm T, Sültmann H, Korf U - Proteome Sci (2010)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Genome Analysis, German Cancer Research Center, Heidelberg, Germany. u.korf@dkfz.de.

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.

No MeSH data available.


Related in: MedlinePlus

Comparing the sensitivity of standard NIR detection and AMSA for endogenous proteins. Detection of beta-Catenin, PDK1, and PCNA in the human colon cancer cell line HT-29 and in the human breast cancer cell lines T47D and MCF7 comparing AMSA with standard NIR. The limit of detection is indicated as red or blue line.
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Figure 5: Comparing the sensitivity of standard NIR detection and AMSA for endogenous proteins. Detection of beta-Catenin, PDK1, and PCNA in the human colon cancer cell line HT-29 and in the human breast cancer cell lines T47D and MCF7 comparing AMSA with standard NIR. The limit of detection is indicated as red or blue line.

Mentions: The general benefit of AMSA for the quantification of low abundance proteins was evaluated further using a set of eleven cancer cell lines. Cell line lysate samples were printed as 14-step serial dilutions and in two subarrays per slide. The first subarray was detected using standard NIR, the other with AMSA. The slides were scanned at different scanner sensitivity settings reflecting the fact that the AMSA detection generally produces stronger signals than the standard NIR detection. Thus, subarray images produced with scanner settings optimal for the AMSA readout, the corresponding subarray of the standard NIR detection was too dim to allow a reasonable comparison between both methods. For this reason, each slide was scanned at different scanner settings and for the direct comparison between standard NIR and AMSA images with comparable signal intensities were chosen (Figure 5). Results indicated that the detection of low abundant proteins indeed benefited from AMSA, as shown for the detection of beta-Catenin and PDK1. The limit of detection increased up to ten dilution steps for beta-Catenin, and PDK1 (Figure 5). However, when calculating the average LOD gain for all eleven cell lines a mean LOD gain of 4- and 5-fold was observed for beta-Catenin, and PDK1, respectively (Figure 6). The detection of abundant proteins such as PCNA did not benefit from AMSA (Figure 5 and 6). Thus, AMSA improved especially the detection of proteins expressed at a low level by reducing the scattering of weak signals and improving signal-to-noise ratios. Antibody-mediated signal amplification strategies promise to be useful for the RPPA-based quantitative analysis of systems-biology type of experiments as well as for the analysis of clinical samples.


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

Brase JC, Mannsperger H, Fröhlich H, Gade S, Schmidt C, Wiemann S, Beissbarth T, Schlomm T, Sültmann H, Korf U - Proteome Sci (2010)

Comparing the sensitivity of standard NIR detection and AMSA for endogenous proteins. Detection of beta-Catenin, PDK1, and PCNA in the human colon cancer cell line HT-29 and in the human breast cancer cell lines T47D and MCF7 comparing AMSA with standard NIR. The limit of detection is indicated as red or blue line.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908584&req=5

Figure 5: Comparing the sensitivity of standard NIR detection and AMSA for endogenous proteins. Detection of beta-Catenin, PDK1, and PCNA in the human colon cancer cell line HT-29 and in the human breast cancer cell lines T47D and MCF7 comparing AMSA with standard NIR. The limit of detection is indicated as red or blue line.
Mentions: The general benefit of AMSA for the quantification of low abundance proteins was evaluated further using a set of eleven cancer cell lines. Cell line lysate samples were printed as 14-step serial dilutions and in two subarrays per slide. The first subarray was detected using standard NIR, the other with AMSA. The slides were scanned at different scanner sensitivity settings reflecting the fact that the AMSA detection generally produces stronger signals than the standard NIR detection. Thus, subarray images produced with scanner settings optimal for the AMSA readout, the corresponding subarray of the standard NIR detection was too dim to allow a reasonable comparison between both methods. For this reason, each slide was scanned at different scanner settings and for the direct comparison between standard NIR and AMSA images with comparable signal intensities were chosen (Figure 5). Results indicated that the detection of low abundant proteins indeed benefited from AMSA, as shown for the detection of beta-Catenin and PDK1. The limit of detection increased up to ten dilution steps for beta-Catenin, and PDK1 (Figure 5). However, when calculating the average LOD gain for all eleven cell lines a mean LOD gain of 4- and 5-fold was observed for beta-Catenin, and PDK1, respectively (Figure 6). The detection of abundant proteins such as PCNA did not benefit from AMSA (Figure 5 and 6). Thus, AMSA improved especially the detection of proteins expressed at a low level by reducing the scattering of weak signals and improving signal-to-noise ratios. Antibody-mediated signal amplification strategies promise to be useful for the RPPA-based quantitative analysis of systems-biology type of experiments as well as for the analysis of clinical samples.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Genome Analysis, German Cancer Research Center, Heidelberg, Germany. u.korf@dkfz.de.

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.

No MeSH data available.


Related in: MedlinePlus