III ProteoRed-ISCII Protein Microarrays Course, 18th-19th October 2012
Antibody colocalization microarrays -a scalable technology for multiplex protein profiling in complex samples-
Mateu Pla-Roca
STANDARDS
Micro and Nanobioengineering Lab Prof. David Juncker
Outline
• Proteins as biomarkers. Written in blood
• Scalability and vulnerability of antibody microarrays (antibody cross-reactivities)
• Antibody colocalization microarrays -the principle-
• Examples
• Concluding remarks
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-The principle- “Every cell in the body leaves a record of it’s physiological state in the products it sheds on the blood” • Tumors “leak” proteins • Tumor interaction with the surrounding cells are mediated
with cytokines, ECM molecules and growing factors. • Tumors induce alterations of surrounding stroma cells and
release proteases that digest normal tissue and plasma proteins.
Proteins as molecular biomarkers –written in blood-
Liotta, L. A. et al Nature 2003
Screening tool based on the quantitative detection molecular biomarkers in blood (or other corporals fluids) is required
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• Interferences limits analytical accuracy for proteins @ pg/ml
N. Leigh Anderson et al. Mol. Cell . Prot. 2002.
Challenge: dynamic range of concentration of 1010
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FDA approved biomarkers
Zhen Zhang and Daniel W. Chan “The Road from Discovery to Clinical Diagnostics: Lessons Learned from the First
FDA-Cleared In Vitro Diagnostic Multivariate Index Assay of Proteomic Biomarkers”
Cancer Epidemiol Biomarkers Prev (2010)
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Biomarker sensitivity & specificity
Polanski and Anderson “ A List of Candidate Cancer Biomarker for Targeted Proteomics “ Biomarker Insights : 2 1– 48 (2006)
Hanash et al. ”Mining the plasma proteome for cancer biomarkers” Nature, 452, (2008)
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Case study for multiplexing: Ovarian cancer
Number of biomarkers
Biomarker Sensitivity Specificity Reference
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Leptin, prolactin OPN, IGF-II
95% 95% Mor et al. 2005
6 Leptin, prolactin
OPN, IGF-II, MIF, CA125 95 % 99 %
Visintin et al. 2008
PANELS OF BIOMARKERS ARE REQUIRED 8
Multiplex sandwich assays (MSAs) as a tool for multiplex protein profiling
Clontech labs
Scalability? 9
Genomics has progressed along an exponential curve Protein profiling has progressed at a much slower pace
DNA microarray (number of spots)
Moore’s law
Microwell plate (number of wells) Antibody microarrays
(number of proteins profiled)
Sequencer (sequences/run)
Scalability: exponential growth over extended periods
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Multiplex sandwich assays (MSAs) as a tool for multiplex protein profiling
High vulnerability 11
Cross-reactivity maps for a 14-plex sandwich assays (MSAs)
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Cross-reactivity scenarios for a multiplex sandwich assays (MSAs)
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Cross-reactivity scenarios for a multiplex sandwich assays (MSAs)
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What to do when you find a cross-reactivity?
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Antibody colocalization microarrays (ACMs) - the principle-
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Antibody colocalization microarrays (ACMs) - new format -
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Antibody colocalization microarrays (ACMs) -technical requirements -
∆x≈ 20 µm
∆y≈ 10 µm
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125 pg/mL
IL-4
IL-6
IL-1
0
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300 m
Antibody colocalization microarrays (ACMs) - example-
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Antibody colocalization microarrays (ACMs) - optimization procedures-
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Antibody colocalization microarrays (ACMs) - optimization procedures-
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Antibody colocalization microarrays (ACMs)
50 proteins (100 Antibodies)
Highly cross-reactive Abs can be used (EGFr and CEA)
Ab pairs from different sources on the same array
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Antibody colocalization microarrays (ACMs) - validation-
vs ELISA vs MSA
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Proteins differentially expressed in the serum of (ER+) breast cancer patients (15) and of healthy controls (11).
Measuring serum samples from breast cancer patients (ER+ subtype)
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Concluding remarks
• Do not blind trust Abs
• You should always screen for possible cross-reactivity sources
• Antibody colocalization microarrays are an alternative to solve this issues. We avoid mixing Abs. Scalability is possible
- Use Abs that cross react
- Compare Abs from distinct sources
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The ACM team: Rym Leulmi, Emmanuel Moreau, Saule Tourekhanova, Wen Luo, Veronique Laforte Huiyan Li
Collaborators: John Bergeron, Michael Hallett, Morag Park and Peter Siegel
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Micro and Nano bioengineering Lab Prof. David Juncker
Pla-Roca, M. et al Molecular &Cellular Proteomics 2012
III ProteoRed-ISCII Protein Microarrays Course, 18th-19th October 2012
STANDARDS
Thanks for your attention!
Questions?
