Integrated microarray solutions in research and diagnostics - Tecan
Transcript of Integrated microarray solutions in research and diagnostics - Tecan
Integrated microarray solutions at the CPGR in Cape Town, South Africa
Reinhard Hiller, PhDDirector
Tecan Workshop12-13 Sep 2007
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
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
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)