How To Develop A Microarray

Integrated microarray solutions at the

CPGR in Cape Town, South Africa

Reinhard Hiller, PhD

Director

[email protected]

Tecan Workshop

CPGR

CPGR

CPGR in a nutshell

CPGR in a nutshell

Service & R&D organisation C P G R b o a rd SOP-based services SOP-based services Collaborative projects Collaborative projects CPGR members ExCo Non-exec directors • Infrastructure • Expertise • Services • Collaborative projects • • InfrastructureInfrastructure • • ExpertiseExpertise • • ServicesServices •

• Collaborative projectsCollaborative projects

Funding CBT, PB; MRC Funding Funding CBT, PB; MRC CBT, PB; MRC Mission

•stimulate excellent research (IP),

•Knowledge base in Genomics & Proteomics

•Boost biotech economy Mission

Mission •

•stimulate excellent research stimulate excellent research (IP),

(IP), •

•Knowledge base in Knowledge base in Genomics & Proteomics Genomics & Proteomics •

Core focus area (e.g. Plant Genomics) Capacity development

CPGR platform perspective

CPGR platform perspective

CPGR: An integrated core technology platform

CPGR: An integrated core technology platform

High -qua lity c usto_{m D} NA micr_oarr ayin g & a_naly sis High -qua lity c usto_{m D} NA micr_oarr ayin g & a naly_sis Ta rget valid ation on ht q RT-P CR p latfo rm & Ta qMan lo w-dens ity arra ys Targ et va lidat ion o n ht q RT-P CR p latfo rm & Ta qMan lo w-dens ity arra ys Affym etrix GeneC hip sy stem for lar ge-sca le genoty ping & RNA expre ssion profili ng Affym etrix GeneC hip sy stem for lar ge-sca le genoty ping & RNA expre ssion profili ng bio com pat ible ht _pr ote in pur ific atio n, M AL DI -TO F/T OF -ba sed pr ote in id & de -no vo s equ enc_ing , ab und anc e & fu nct_ion al pro tein _m icr_oar ray_s bio com pat ible ht _pr ote in pur ific atio n, M AL DI -TO F/T OF -ba sed pr ote in id & de -no vo s equ enc_ing , ab und anc e & fu nct_ion al pro tein _m icr_oar ray_s

Project / service pipeline: July 2007

Project / service pipeline: July 2007

Concept Preparation In progress Finished

D is c o n ti n u e d 23% 5% 20% 48% 4% Preparation Concept In progress Finished Disco ntinued Biomedical Plant Biotech Bioprospect. Veterinary n=19 n=39 n=16 n=4

Tb 15% Clinical trial 0% Various 11% Cancer 15% Malaria 9% Biomarker_9% Diagnostics 13% Drug discovery 4% Asthma 4% Yeast 6% Parasites 2% NAFLD 2% Mouse 2% Maize 2% HIV 2% CVD 2% Grape 2% Pharmaceutical 0% Clinical trial Pharmaceutical Grape CVD HIV Maize Mouse NAFLD Parasites Yeast Asthma Diagnostics Drug discovery Biomarker Malaria Various Cancer Tb

Project / service pipeline: July 2007

Workflows

Workflows

Analytical work-flows in GENOMICS

Analytical work-flows in GENOMICS

RNA expression & DNA genotyping microarrays

RNA expression & DNA genotyping microarrays Affymetrix GeneChip platform Affymetrix GeneChip platform Other customized commercial arrays, e.g., Agilent Other customized commercial arrays, e.g., Agilent Custom oligo- & cDNA manufactur -ing Custom oligo- & cDNA manufactur -ing

Discovery

• High genome coverage

• High quality standards (e.g., Agilent bioanalyser)

• High reliability (e.g., Tecan hyb station)

Custom microarrays & ABI 7900 TaqMan system Custom microarrays & ABI

7900 TaqMan system Custom DNA microarrays Custom DNA microarrays TaqMan RT-PCR & genotyping & miRNA assays

Validation

• Focussed validation of large-scale genomic / transcriptomic

data

• Low-density arrays and / or RT-PCR assays

• High throughput

• Large sample numbers

• High quality

• Low sample requirement

CGH

SNPs

Chromosome Gene mRNA Exons µRNAs

Copy-number

analysis Genotyping _regulationGene Differential _{splicing regulation}RNA

ChIP

on

chip

DNA

RNA

• AffyAffy ••ExiqonExiqon • • BioPlexBioPlex • • ABI 7900ABI 7900 • • Affy Affy • • AgilentAgilent

DNA microarray applications at CPGR

(DNA) microarray workflows at CPGR

(DNA) microarray workflows at CPGR

Liquid-handling robot

Spotter

Hyb station

Scanner

•

Through-put

•

Cost-efficiency

•

Quality

Sample Processing Chromatography Electrophoresis • Separation • Identification • Quantification • Fractionation • Concentration Digestion Shotgun • MS/MS • iTRAQ • iCAT • MS/MS • Peptide IDs • Sequence

Peaks / proteins of interest

1 D / 2 D H P L C M A L D I -T O F / T O F Extraction/ purification Protein profiling/Imaging Mass Spectrometry (IMS)

Integrated Proteomics at CPGR

Integrated Proteomics at CPGR

A ll e rg e n A A ll e rg e n B A ll e rg e n C A ll e rg e n D Asthm a Rhinitis Eczem a 0 10 20 30 40 50 60

Protein microarray development

Protein microarray development

Extract Extract

Side-step: Decreased costs

via

increased throughput

Side-step: Decreased costs

via

increased throughput

• 36 Slides á 4 assays á 80 allergens • = 144 IgE assays per run

• = > 10.000 reactions per run • IgE & IgG (IgG4)

• = > 20.000 reactions per run

• 4 Slides á 16 assays á 80 allergens • = 64 assays per run (= 4 slides) • = > 5.100 reactions per batch • IgE & IgG (IgG4)

• = > 10.200 reactions per batch 4 assays per slide

4 assays per slide

36 slides per run

Examples

Examples

Biochip: Modified glass slide (25x75 mm) Biochip: Modified glass

slide (25x75 mm)

4 reaction sites (3 samples + 1 control)

4 reaction sites (3 samples + 1 control) (To date) 80 allergens

microarrayed in each reaction site (4 identical

copies) (To date) 80 allergens

microarrayed in each reaction site (4 identical

copies)

Allergen molecule Allergen molecule

IgE-antibdy (in patient‘s-serum)

IgE-antibdy (in patient‘s-serum) Fluorescence-labelled secondary anti-human IgE antibody Fluorescence-labelled secondary anti-human IgE antibody Allergen-triplet after fluorescence-reaction Allergen-triplet after fluorescence-reaction

Sample: Venous / capillary serum

Sample: Venous

/ capillary serum Incubate SampleIncubate Sample

Manual allergen microarray assay

Manual allergen microarray assay

Incubate Secondary Antibody Incubate Secondary Antibody Prepare Allergen Microarray Wash Allergen Microarray Scan Microarray(s) Scan Microarray(s) Wash Allergen Microarray Analyse Microarray Image(s) Analyse Microarray Image(s) Duration: 3 h Duration: 3 h Duration: 10‘ Duration: 10‘

Multiplex diagnostics: Component-resolved diagnosis

IgE profiles in asthmatic children (2006)

IgE profiles in asthmatic children (2006)

(Allergen) microarray assay automation

(Allergen) microarray assay automation

•

Diagnostics

•

Epidemiological studies

•

R&D (profiling)

•

(Diagnostics)

•

R&D

•

Validation

(Seafood) allergen characterisation

(Seafood) allergen characterisation

Allergen discovery Allergen discovery Allergen profiling

Allergen profiling Diagnostics_Diagnostics

Reactive bands Reactive bands MTP MTP Beads_Beads Immuno-blots Immuno-blots Affinity capturing Affinity capturing Cell culture Cell culture HPLC HPLC MS MS Microarrays Microarrays Columns Columns Allergen detetection Ab assays for allergen detection (bead assays)… Allergen detetection Ab assays for allergen detection (bead assays)…

New (and expected) allergens in seafood species

New (and expected) allergens in seafood species

Diagnostic assays for workers in the seafood industry -> better prevention & treatment of occupational allergies Diagnostic assays for workers in the seafood industry

-> better prevention & treatment of

occupational allergies

Extraction Allergenic molecule Non-Allergenic molecule Fractionation

Array-based allergen profiling

Array-based allergen profiling

6 mm

6

m

Identification / characterization of latex allergens

Identification / characterization of latex allergens

Identification / characterization of latex allergens

• Screening with 200 samples from latex allergic patients • Pearson correlation (> 0.85, p > 0.05) • Pre-characterize unknown fractions Information gained: • Pre-characterized allergensbased on cross-reactivity towards homologous proteins • Information about composition of

fractionsplus “state of allergens” = important for manufacturers of immunotherapy reagents (extracts)

Allergen microarrays & immunotherapy development

Allergen microarrays & immunotherapy development

Allergen concentration in solution

Ig E c o n c e n tr a ti o n ( F I v a lu e s )

Which biological source contains the immunodominant allergen

suitable for immunotherapy?

Which biological source contains the immunodominant allergen

0 10000 20000 30000 40000 50000 60000 70000 1 2 3 4 5 6 7 8 9 10 nOle e 2 rBet v 2 rCyn d 12 rHel a 2 rHev b 8 rMer a 1 rPar j 3 rPhl p 12 rPho d 2 40 µg – 5 pg / ml 65 ng / ml

Screening for immunodominant allergens & epitope profiling

Screening for immunodominant allergens & epitope profiling

Harwanegg & Hiller, unpublished results

Bet v 2 (birch profilin) contains all the

epitopes of 8 homologous profilins

Bet v 2 (birch profilin) contains all the

epitopes of 8 homologous profilins Therapy candidate Therapy candidate

Microarray-based B-cell epitope profiling: Tb peptide arrays

Microarray-based B-cell epitope profiling: Tb peptide arrays

Protein a, b, c Protein a, b, c…… Peptide synthesis Peptide synthesis Peptide microarray Peptide microarray • Linear ab binding epitopes • Diagnostic peptides / signatures • Therapeutic targets •

• Linear abLinear ab binding binding epitopes

epitopes •

• Diagnostic peptides / Diagnostic peptides / signatures

signatures •

• Therapeutic targetsTherapeutic targets

Whole proteome

Manual vs automated Tb peptide arrays

Manual vs automated Tb peptide arrays

Manual Automated A u to m a te d M a n u a l

Increase in foreground signal using HSPro4800 2 slide batches 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0

F635 Median - B635 F635 Total Intensity

v a lu e s e x p re s s e d a s p e rc e n ta g e s batch 793 manual batch 793 HS4800Pro batch 866 manual batch 866 HS4800Pro

Significant increase in S/N in automated assays

Significant increase in S/N in automated assays

Signal - background Total signal

Increase in the proportion of spots detected above cut-off levels

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% % Spots with SNR>3 batch 866 HS4800Pro batch 866 manual batch 793 HS4800Pro batch 793 manual

% spots above cut-off

•Secondary Antibody •Incubation

•Precision (within & between slide & between runs, batch-to-batch etc)

Other microarray applications

Other microarray applications

• Operon

• Dual malaria parasite DNA microarrays (HS 4800) • Agilent

• 1-plex and 4-plex genotyping arrays (CGH!) (HS 4800) • Exiqon

• MirCury miRNA arrays -> miRNA biomarker focus area in South Africa (HS 4800) • JPT

• Tb peptide arrays; CMV arrays; infectious disease arrays (malaria); allergen epitope profiling (HS 4800)

• VBC-GENOMICS

• Allergen microarrays (HS 4800) • Gentel

• Cytokine antibody arrays (Hydroflex); cancer biomarker arrays (HS 4800) • CPGR

• Arrays of fractionated Tb cultures; tissue extract arrays; functional protein arrays (p450, p53) (HS 4800 & HydroFlex)

Translational issues

Translational issues

Signature

Signature

Biology

Biology

Pattern

_Pattern

Better diagnostics Better diagnostics

Novel drug targets Novel drug targets

Novel therapeutic applications Novel therapeutic applications More efficient treatments More efficient treatments Personalized medicine Personalized medicine

Early discovery / pre-clinical development: min. 2 years

Biomarker/MDx development

Biomarker/MDx development

GAP

_GAP

Acknowledgements

Acknowledgements

Companies

• JPT (Germany)

• VBC-GENOMICS (Austria)

• Christian Harwanegg

CPGR

•

Jonathan Blackburn

• Anne Brill

•

Rachel van Dyk

• Jo McBride

• Maré Vlok

• Kutala Pangwa

Funding

•Cape Biotech Trust (CBT) •PlantBio (PB)

•Medical Research Council (MRC)

Scientists

• Mark Bowmaker (SA) • Mohamed Jeebhay (SA) • Andreas Lopata (SA) • Rajen Naidoo (SA)