R&D - Tecan
R&D - Tecan
R&D - Tecan
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
Integrated microarray solutions at the<br />
CPGR in Cape Town, South Africa<br />
Reinhard Hiller, PhD<br />
Director<br />
Reinhard.hiller@cpgr.org.za<br />
<strong>Tecan</strong> Workshop<br />
12-13 Sep 2007
CPGR
CPGR in a nutshell<br />
CPGR<br />
members<br />
Collaborative<br />
projects<br />
Service & R&D<br />
organisation<br />
Non-exec<br />
directors<br />
ExCo<br />
CPGR board<br />
SOP-based<br />
services<br />
• Infrastructure<br />
• Expertise<br />
• Services<br />
• Collaborative projects<br />
Funding<br />
CBT, PB; MRC<br />
Mission<br />
•stimulate stimulate excellent research<br />
(IP),<br />
•Knowledge Knowledge base in<br />
Genomics & Proteomics<br />
•Boost Boost biotech economy
CPGR platform perspective<br />
Capacity<br />
development<br />
Core focus<br />
area (e.g.<br />
Plant<br />
Genomics)<br />
New<br />
Commercial<br />
ventures<br />
Collaborations
CPGR: An integrated core technology platform<br />
Affymetrix<br />
Affymetrix<br />
GeneChip GeneChip system system<br />
for large-scale<br />
large-scale<br />
genotyping genotyping & & RNA RNA<br />
expression expression profiling profiling<br />
High-quality High-quality custom custom DNA DNA<br />
microarraying microarraying & & analysis analysis<br />
biocompatible biocompatible ht ht protein protein<br />
purification, purification, MALDI- MALDI-<br />
TOF/TOF-based TOF/TOF-based protein protein<br />
id id & & de-novo de-novo sequencing,<br />
sequencing,<br />
abundance abundance & & functional functional<br />
protein protein microarrays<br />
microarrays<br />
Target Target validation validation on on<br />
ht ht qRT-PCR qRT-PCR platform platform<br />
& & TaqMan TaqMan low-density<br />
low-density<br />
arrays arrays
Project / service pipeline: July 2007<br />
Concept Preparation In progress Finished<br />
n=19 n=39 n=16 n=4<br />
20%<br />
23%<br />
5%<br />
4%<br />
48%<br />
Discontinued<br />
Biomedical<br />
Plant Biotech<br />
Bioprospect.<br />
Veterinary<br />
Preparation<br />
Concept<br />
In progress<br />
Finished<br />
Disco ntinued
Project / service pipeline: July 2007<br />
Cancer<br />
15%<br />
Clinical trial<br />
0%<br />
Pharmaceutical<br />
0%<br />
Tb<br />
15%<br />
Various<br />
11%<br />
Malaria<br />
9%<br />
Grape<br />
2%<br />
CVD<br />
2%<br />
HIV<br />
2%<br />
Biomarker<br />
9%<br />
Maize<br />
2%<br />
Mouse<br />
2%<br />
NAFLD<br />
2%<br />
Parasites<br />
2% Yeast<br />
6%<br />
Asthma<br />
4%<br />
Diagnostics<br />
13%<br />
Drug discovery<br />
4%<br />
Clinical trial<br />
Pharmaceutical<br />
Grape<br />
CVD<br />
HIV<br />
Maize<br />
Mouse<br />
NAFLD<br />
Parasites<br />
Yeast<br />
Asthma<br />
Diagnostics<br />
Drug discovery<br />
Biomarker<br />
Malaria<br />
Various<br />
Cancer<br />
Tb
Workflows
Analytical work-flows in GENOMICS<br />
RNA expression & DNA genotyping<br />
microarrays<br />
Affymetrix<br />
GeneChip<br />
platform<br />
• High-density arrays<br />
• Sequenced organisms<br />
Discovery<br />
Other<br />
customized<br />
commercial<br />
arrays, e.g.,<br />
Agilent<br />
Custom<br />
oligo- &<br />
cDNA<br />
manufactur<br />
-ing<br />
• Non-model<br />
organisms<br />
cDNA arrays<br />
• Custom<br />
development<br />
• High genome coverage<br />
• High quality standards (e.g., Agilent bioanalyser)<br />
• High reliability (e.g., <strong>Tecan</strong> hyb station)<br />
Custom microarrays & ABI<br />
7900 TaqMan system<br />
Custom DNA<br />
microarrays<br />
Validation<br />
TaqMan RT-<br />
PCR &<br />
genotyping<br />
& miRNA<br />
assays<br />
• Focussed validation of largescale<br />
genomic / transcriptomic<br />
data<br />
• Low-density arrays and / or<br />
RT-PCR assays<br />
• High throughput<br />
• Large sample numbers<br />
• High quality<br />
• Low sample requirement<br />
• Low costs
DNA microarray applications at CPGR<br />
CGH SNPs<br />
Gene<br />
expression<br />
Splice<br />
variants<br />
µRNAs<br />
Chromosome Gene Promoters mRNA Exons µRNAs<br />
Copy-number<br />
analysis<br />
• Affy<br />
• Agilent<br />
• NimbleGen<br />
• Operon<br />
DNA RNA<br />
Genotyping<br />
• Affy<br />
• Agilent<br />
• ABI 7900<br />
ChIP<br />
on<br />
chip<br />
Regulatory<br />
assessment<br />
• Affy<br />
• Agilent<br />
Gene<br />
regulation<br />
• Affy<br />
• Agilent<br />
• Operon<br />
• ABI 7900<br />
Differential<br />
splicing<br />
RNA<br />
regulation<br />
• Affy • Exiqon<br />
• BioPlex<br />
• ABI 7900
(DNA) microarray workflows at CPGR<br />
Liquid-handling robot Spotter Hyb station Scanner<br />
• Through-put<br />
• Cost-efficiency<br />
• Quality<br />
• Compatibility with other (microarray) applications
Integrated Proteomics at CPGR<br />
Electrophoresis<br />
• Separation<br />
• Identification<br />
• Quantification<br />
Protein expression<br />
• MS/MS<br />
• iTRAQ<br />
• iCAT<br />
Sample<br />
Processing<br />
Digestion<br />
Chromatography<br />
m/z<br />
Chromatography<br />
• Fractionation<br />
• Concentration<br />
• MS/MS<br />
Shotgun<br />
• Peptide IDs<br />
• Sequence<br />
Peaks / proteins of interest<br />
Extraction/<br />
purification<br />
1D/2D HPLC<br />
MALDI-TOF/TOF<br />
Protein<br />
profiling/Imaging Mass<br />
Spectrometry (IMS)<br />
Protein<br />
microarrays<br />
Suspension<br />
arrays
Protein microarray development<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
Allergen A<br />
Allergen B<br />
Allergen C<br />
Allergen D<br />
Eczema<br />
Rhinitis<br />
Asthma<br />
Extract Component(s) Component(s<br />
Component(s)<br />
Microarray (Diagnostic) profile
Side-step: Decreased costs via via increased throughput<br />
• 36 Slides á 4 assays á 80 allergens<br />
• = 144 IgE assays per run<br />
• = > 10.000 reactions per run<br />
• IgE & IgG (IgG4)<br />
• = > 20.000 reactions per run<br />
4 assays per slide<br />
36 slides per run<br />
• 4 Slides á 16 assays á 80 allergens<br />
• = 64 assays per run (= 4 slides)<br />
• = > 5.100 reactions per batch<br />
• IgE & IgG (IgG4)<br />
• = > 10.200 reactions per batch
Examples
Allergen-triplet after<br />
fluorescence-reaction<br />
(To date) 80 allergens<br />
microarrayed in each<br />
reaction site (4 identical<br />
copies)<br />
IgE-antibdy (in patient‘sserum)<br />
Allergen molecule<br />
Biochip: Modified glass<br />
slide (25x75 mm)<br />
4 reaction sites (3<br />
samples + 1 control)<br />
Fluorescence-labelled<br />
secondary anti-human<br />
IgE antibody
Manual allergen microarray assay<br />
Sample: Venous<br />
/ capillary serum<br />
Wash Allergen<br />
Microarray<br />
Prepare Allergen<br />
Microarray<br />
Scan<br />
Microarray(s)<br />
Incubate Sample<br />
Duration: 10‘<br />
Wash Allergen<br />
Microarray<br />
Duration: 3 h<br />
Analyse<br />
Microarray<br />
Image(s)<br />
Incubate<br />
Secondary<br />
Antibody
Multiplex diagnostics: Component-resolved diagnosis
IgE profiles in asthmatic children (2006)<br />
Hiller, Jeebhay, Lopata & Naidoo, manuscript in preparation
(Allergen) microarray assay automation<br />
• Diagnostics<br />
• Epidemiological studies<br />
• R&D (profiling)<br />
• (Diagnostics)<br />
• R&D<br />
• Validation
(Seafood) allergen characterisation<br />
Allergen profiling Allergen discovery<br />
Diagnostics<br />
Allergen<br />
detetection<br />
Ab assays for<br />
allergen detection<br />
(bead assays)…<br />
Reactive<br />
bands<br />
Immunoblots<br />
Affinity<br />
capturing<br />
MTP Beads<br />
MS<br />
Microarrays<br />
HPLC<br />
Columns<br />
New (and expected) allergens in<br />
seafood species<br />
Cell culture<br />
Diagnostic assays<br />
for workers in the<br />
seafood industry -<br />
> better prevention<br />
& treatment of<br />
occupational<br />
allergies
Array-based allergen profiling<br />
Extraction<br />
Fractionation<br />
Allergenic<br />
molecule<br />
Non-Allergenic<br />
molecule<br />
6 mm<br />
6 mm
Identification / characterization of latex allergens<br />
• Screening with 200<br />
samples from latex<br />
allergic patients<br />
• Pearson correlation<br />
(> 0.85, p > 0.05)<br />
• Pre-characterize<br />
unknown fractions<br />
Information gained:<br />
• Pre-characterized<br />
allergens based on<br />
cross-reactivity<br />
towards homologous<br />
proteins<br />
• Information about<br />
composition of<br />
fractions plus “state of<br />
allergens” = important<br />
for manufacturers of<br />
immunotherapy<br />
reagents (extracts)
Allergen microarrays & immunotherapy development<br />
IgE concentration (FI values)<br />
Allergen concentration in solution<br />
Which biological source contains<br />
the immunodominant allergen<br />
suitable for immunotherapy?
Screening for immunodominant allergens & epitope profiling<br />
70000<br />
60000<br />
50000<br />
40000<br />
30000<br />
20000<br />
10000<br />
0<br />
65 ng / ml<br />
1 2 3 4 5 6 7 8 9 10<br />
40 µg – 5 pg / ml<br />
Harwanegg & Hiller, unpublished results<br />
nOle e 2<br />
rBet v 2<br />
rCyn d 12<br />
rHel a 2<br />
rHev b 8<br />
rMer a 1<br />
rPar j 3<br />
rPhl p 12<br />
rPho d 2<br />
Bet v 2 (birch profilin)<br />
contains all the<br />
epitopes of 8<br />
homologous profilins<br />
Therapy<br />
candidate
Microarray-based B-cell epitope profiling: Tb peptide arrays<br />
Peptide synthesis<br />
Peptide microarray<br />
Whole proteome<br />
Protein a, b, c… c<br />
• Linear ab binding<br />
epitopes<br />
• Diagnostic peptides /<br />
signatures<br />
• Therapeutic targets
Manual vs automated Tb peptide arrays<br />
Manual Automated<br />
Manual<br />
Automated
values expressed as percentages<br />
Significant increase in S/N in automated assays<br />
100.0<br />
90.0<br />
80.0<br />
70.0<br />
60.0<br />
50.0<br />
40.0<br />
30.0<br />
20.0<br />
10.0<br />
0.0<br />
Increase in foreground signal using HSPro4800<br />
2 slide batches<br />
F635 Median - B635 F635 Total Intensity<br />
Signal - background Total signal<br />
batch 793 manual<br />
batch 793 HS4800Pro<br />
batch 866 manual<br />
batch 866 HS4800Pro<br />
• Secondary Antibody<br />
• Incubation<br />
• Precision (within & between slide & between runs,<br />
batch-to-batch etc)<br />
100%<br />
• Specificity (comp. inhibition assays)<br />
90%<br />
80%<br />
70%<br />
60%<br />
50%<br />
40%<br />
30%<br />
20%<br />
10%<br />
0%<br />
Increase in the proportion of spots detected above cut-off levels<br />
% Spots with SNR>3<br />
% spots above cut-off<br />
batch 866 HS4800Pro<br />
batch 866 manual<br />
batch 793 HS4800Pro<br />
batch 793 manual
Other microarray applications<br />
• Operon<br />
• Agilent<br />
• Exiqon<br />
• JPT<br />
• Dual malaria parasite DNA microarrays (HS 4800)<br />
• 1-plex and 4-plex genotyping arrays (CGH!) (HS 4800)<br />
• MirCury miRNA arrays -> miRNA biomarker focus area in South Africa (HS 4800)<br />
• Tb peptide arrays; CMV arrays; infectious disease arrays (malaria); allergen epitope profiling (HS<br />
4800)<br />
• VBC-GENOMICS<br />
• Gentel<br />
• CPGR<br />
• Allergen microarrays (HS 4800)<br />
• Cytokine antibody arrays (Hydroflex); cancer biomarker arrays (HS 4800)<br />
• Arrays of fractionated Tb cultures; tissue extract arrays; functional protein arrays (p450, p53) (HS<br />
4800 & HydroFlex)
Translational issues<br />
Biomarker/MDx development GAP<br />
Biology Pattern<br />
Early discovery / pre-clinical development: min. 2 years<br />
Signature<br />
Personalized<br />
medicine<br />
Validation<br />
Better diagnostics<br />
Novel drug targets<br />
Novel therapeutic<br />
applications<br />
More efficient<br />
treatments
Acknowledgements<br />
CPGR<br />
• Jonathan Blackburn<br />
• Anne Brill<br />
• Rachel van Dyk<br />
• Jo McBride<br />
• Maré Vlok<br />
• Kutala Pangwa<br />
Scientists<br />
• Mark Bowmaker (SA)<br />
• Mohamed Jeebhay (SA)<br />
• Andreas Lopata (SA)<br />
• Rajen Naidoo (SA)<br />
Companies<br />
• JPT (Germany)<br />
• VBC-GENOMICS (Austria)<br />
Funding<br />
• Christian Harwanegg<br />
•Cape Biotech Trust (CBT)<br />
•PlantBio (PB)<br />
•Medical Research Council (MRC)
Change language