Integrated microarray solutions at the CPGR in Cape Town, South Africa

Reinhard Hiller, PhDDirector

Reinhard.hiller@cpgr.org.za

Tecan Workshop12-13 Sep 2007

CPGRCPGRCPGR

CPGR in a nutshellCPGR in a nutshell

Service & R&D organisation

CP

GR

boa

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

FundingFunding

CBT, PB; MRCCBT, PB; MRC

Mission

•stimulate excellent research (IP),

•Knowledge base in Genomics & Proteomics

•Boost biotech economy

MissionMission

••stimulate excellent research stimulate excellent research

(IP), (IP),

••Knowledge base in Knowledge base in

Genomics & ProteomicsGenomics & Proteomics

••Boost biotech economyBoost biotech economy

Core focus area (e.g.

Plant Genomics)

Capacity development

CPGR platform perspectiveCPGR platform perspective

New Commercial

ventures

Collaborations

CPGR: An integrated core technology platformCPGR: An integrated core technology platform

High-quality custom DNA

microarraying & analysis

High-quality custom DNA

microarraying & analysis

Target validation on

ht qRT-PCR platform

& TaqMan low-density

arrays

Target validation on

ht qRT-PCR platform

& TaqMan low-density

arrays

Affym

etrix

GeneC

hip system

for large-scale

genotyping & RN

A

expression profiling

Affym

etrix

GeneC

hip system

for large-scale

genotyping & RN

A

expression profiling

biocompatible ht protein

purification, MALDI-

TOF/TOF-based protein

id & de-novo sequencing,

abundance & functional

protein microarrays

biocompatible ht protein

purification, MALDI-

TOF/TOF-based protein

id & de-novo sequencing,

abundance & functional

protein microarrays

Project / service pipeline: July 2007Project / service pipeline: July 2007

Concept Preparation In progress Finished

Discontinued

23%

5%

20%

48%

4%

Preparation

Concept

In progress

Finished

Disco ntinued

Biomedical

Plant Biotech

Bioprospect.

Veterinaryn=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 2007Project / service pipeline: July 2007

WorkflowsWorkflowsWorkflows

Analytical work-flows in GENOMICSAnalytical 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-density arrays• Sequenced organisms

• Non-modelorganismscDNA arrays• Customdevelopment

• 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 & miRNAassays

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 • Low costs

CGH SNPsGene

expression

Splicevariants µRNAs

Chromosome Gene mRNA Exons µRNAs

Copy-numberanalysis

Genotyping Gene regulation

Differential splicing

RNA regulation

ChIP

on

chip

Promoters

Regulatory assessment

DNA RNA

•• AffyAffy

•• AgilentAgilent

•• NimbleGenNimbleGen

•• OperonOperon

•• AffyAffy

•• AgilentAgilent

•• ABI 7900ABI 7900

•• AffyAffy

•• AgilentAgilent

•• OperonOperon

•• ABI 7900ABI 7900

•• AffyAffy •• ExiqonExiqon

•• BioPlexBioPlex

•• ABI 7900ABI 7900

•• Affy Affy

•• AgilentAgilent

DNA microarray applications at CPGRDNA 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• Compatibility with other (microarray) applications

Sample

Processing

ChromatographyElectrophoresis

• Separation

• Identification

• Quantification

• Fractionation

• Concentration

Digestion

Shotgun

• MS/MS

• iTRAQ

• iCAT

• MS/MS

• Peptide IDs

• Sequence

Peaks / proteins of interest

1D

/2D

HP

LC

MA

LD

I-T

OF/TO

F

Extraction/purification

Protein profiling/Imaging Mass Spectrometry (IMS)

Integrated Proteomics at CPGRIntegrated Proteomics at CPGR

m/z

Protein expression

Protein microarrays

Suspension arrays

Chromatography

Allerg

en

A

Allerg

en

B

Allerg

en

C

Allerg

en

D

Asthma

RhinitisEczema

0

10

20

30

40

50

60

Protein microarray developmentProtein microarray development

ExtractExtractExtract Component(s)Component(sComponent(s)) MicroarrayMicroarrayMicroarray (Diagnostic) profile(Diagnostic) profile(Diagnostic) profile

Side-step: Decreased costs via increased throughputSide-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 slide4 assays per slide

36 slides per run36 slides per run

ExamplesExamplesExamples

Biochip: Modified glassslide (25x75 mm)

Biochip: Modified glassslide (25x75 mm)

4 reaction sites (3 samples + 1 control)

4 reaction sites (3 samples + 1 control)

(To date) 80 allergensmicroarrayed in eachreaction site (4 identical

copies)

(To date) 80 allergensmicroarrayed in eachreaction site (4 identical

copies)

Allergen moleculeAllergen molecule

IgE-antibdy (in patient‘s-serum)

IgE-antibdy (in patient‘s-serum)

Fluorescence-labelledsecondary anti-human

IgE antibody

Fluorescence-labelledsecondary anti-human

IgE antibody

Allergen-triplet afterfluorescence-reaction

Allergen-triplet afterfluorescence-reaction

Sample: Venous / capillary serum

Sample: Venous / capillary serum

Incubate SampleIncubate Sample

Manual allergen microarray assayManual 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)

AnalyseMicroarray Image(s)

Duration: 3 hDuration: 3 h

Duration: 10‘Duration: 10‘

Multiplex diagnostics: Component-resolved diagnosis Multiplex diagnostics: Component-resolved diagnosis

IgE profiles in asthmatic children (2006)IgE profiles in asthmatic children (2006)

Hiller, Jeebhay, Lopata & Naidoo, manuscript in preparation

(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 discoveryAllergen discoveryAllergen profilingAllergen profiling DiagnosticsDiagnostics

Reactive bands

Reactive bands

MTPMTP BeadsBeads

Immuno-blots

Immuno-blots

Affinity capturing

Affinity capturing

Cell cultureCell culture

HPLCHPLC

MSMS

MicroarraysMicroarrays

ColumnsColumns

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 profilingArray-based allergen profiling

6 mm

6 mm

Identification / characterization of latex allergensIdentification / characterization of latex allergensIdentification / 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 allergens based on cross-reactivity towards homologous proteins

• Information about composition of fractions plus “state of allergens” = important for manufacturers of immunotherapy reagents (extracts)

Allergen microarrays & immunotherapy developmentAllergen microarrays & immunotherapy development

Allergen concentration in solution

IgE

co

ncen

trati

on

(F

I valu

es)

Which biological source contains the immunodominant allergen suitable for immunotherapy?

Which biological source contains the immunodominant allergen suitable for immunotherapy?

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 profilingScreening 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 arraysMicroarray-based B-cell epitope profiling: Tb peptide arrays

Protein a, b, cProtein a, b, c……

Peptide synthesisPeptide synthesis

Peptide microarrayPeptide microarray

• Linear ab binding epitopes

• Diagnostic peptides / signatures

• Therapeutic targets

•• Linear Linear abab binding binding

epitopesepitopes

•• Diagnostic peptides / Diagnostic peptides /

signaturessignatures

•• Therapeutic targetsTherapeutic targets

Whole proteomeWhole proteome

Manual vs automated Tb peptide arraysManual vs automated Tb peptide arrays

Manual Automated

Au

tom

ate

dM

an

ua

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

va

lue

s e

xp

res

se

d a

s p

erc

en

tag

es

batch 793 manual

batch 793 HS4800Pro

batch 866 manual

batch 866 HS4800Pro

Significant increase in S/N in automated assaysSignificant 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)

• Specificity (comp. inhibition assays)

Other microarray applicationsOther 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 issuesTranslational issues

SignatureSignatureBiologyBiology PatternPattern

Better diagnosticsBetter diagnostics

Novel drug targetsNovel 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 developmentBiomarker/MDx development GAPGAP

Validation

AcknowledgementsAcknowledgements

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)