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?