Post on 21-Jun-2020
“Multiplex Immunoassay Development and Validation using Ultra-thin Film Nitrocellulose."
Robert Negm, PhDVice President, GenTel BioSciences
TECAN SYMPOSIUM 2007
2
Protein Array Variety• Quantitative Multiplex Immunoassays
– Cytokine, Metabolic, COAG– PhosphoArrays
• Single Capture Antibody Arrays
• Antigen Arrays– Serological Arrays– Ab Specificity Screening– Kinase Substrate Profiling/Peptide arrays
• Reverse Western/Lysate arrays
3
PATH™Protein
Microarray Slide
Conventional Nitrocellulose
Slide
6.07
3.07
% CV
500
500
Sample Size
24998
398
Mean RFU
1517
15
Std. Dev.
Conventional Nitrocellulose
PATH™ Slide
Fluorescence Background ComparisonPATH™ vs Conventional Nitrocellulose Slide
Blank slides scanned at identical parameters. 543 nm excitation (Cy3). ScanArray® 4000 and QuantArray® software. 500 evenly spaced 500 µm circles. PMT = 90, Gain = 75.
PATH Performance
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Traditional Blood Analyte Measurement
Cy3 Labeled Streptavidin
Biotin Labeled Secondary Ab
Antigen (Cytokine)
Primary Ab
Cytokine Sandwich ImmunoassayAntibody Array
5
10
100
1000
10000
100000
0.1 1 10 100 1000 10000 100000Antigen Concentration (pg/mL)
Sign
al (R
FU)
IL-1 BetaIL-2IL-6TNF-AlphaLOD
0.5 pg/mL 5 pg/mL 50 pg/mL 500 pg/mL 5 ng/mL 50 ng/mL
IL-1 Beta
IL-2
IL-6
TNF-Alpha
5 Logs Dynamic Range
Ambient analyte theory combined with fluoresence.
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Cytokine Sandwich ImmunoassayHead-to-head Signal-to-Noise Comparison
PATH Performance
7
QA/QC
• Nitrate composition
• Uniform thickness
• Hydrophobicity and contact angle
• Protein binding capacity
8
Biomarker Profiling Drivers• More data with less sample in less time
• Multiple analytes improve clinical sensitivity and specificity in disease diagnostics
• Early detection, benefits of therapy and liklihood of disease recurrence
• Surrogate endpoints better than clinical endpoints
9
Arraying & Imaging
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PATH™ HTS
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APiX™ HTS• Glass-bottom 3x5 Microplate - design
• Wells facing up
• Wells facing down
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Glass-bottom 3x5 Microplate - Assembly
13
Quantitative Multiplex Immunoassays
Analyte
Detection antibody
Capture antibody
Analyte
Detection antibody
Capture antibody
Analyte
Detection antibody
Capture antibody
14
GenTel Assay Validation
Methods– Cross-reactivity– Dilution Optimization– Standard Curve Validation– Assay Precision– Dilutional Recovery
15
Eliminating Cross Talk
a) Nonspecific binding between the detector antibodies antigen-capture antibody. b) Nonspecific binding between the detector antibody with capture antibody. c) Probing microarrays of capture antibodies with individual antigens.d) Probing microarrays of capture antibodies with cocktails of antigens.
16
Protein Liquid Handling Process Workflow
Making standard and samples dilutions
Arranging standards and samples on a source plate
Transferring standards and samples from a source plate to chips
Adding detection antibody and dye Incubation Washing
17
s
Image from an Assay Platefour Chips with 64 Chambers
18
Printing format improvement:reduce variations with scattered
replicatesRandomized Scattered ArrayLinear Array
19
Robotic assay and normalization protocol plying normalization (reduced variability)
Analyte = IL-06 (554543) 0.125
log10(Conc)
logS
igna
l
1.0 1.5 2.0 2.5 3.0 3.5
2.5
3.0
3.5
4.0
4.5
No normalized
1.0 1.5 2.0 2.5 3.0 3.5
3.0
3.5
4.0
4.5
5.0
normalization
Chip12
34
Before normalization After normalization
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Dilutional RecoveryTNFb (551222)
Concentration
Pro
b
0.0
0.2
0.4
0.6
0.8
1.0
0.1 0.5 1 5 10 50 100 500 1000
4C:90-min_1
CVLLOQULOQ
9 % 2.21771.5
TRAIL (550517)
Concentration
Pro
b
0.0
0.2
0.4
0.6
0.8
1.0
0.1 0.5 1 5 10 50 100 500 1000 5000
4C:90-min_1
CVLLOQULOQ
9.8 % 103765
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Five (5) Quantitative Multiplex Immunoassays
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Cytokine Measurements in Serum, Plasma or Cell Lysates
GM-CSFIFNγIL-1βIL-2IL-3IL-4IL-5IL-6IL-7IL-8
IL-10IL-12IL-13
MCP-1TNFαTNFβVEGF
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Human Cytokine Chip Data
24
Dose-Response curves
log10(Conc)
logS
igna
l
2
3
4
0 1 2 3 4
GM-CSF IFNg
0 1 2 3 4
IL-01b IL-02
0 1 2 3 4
IL-03 IL-04
IL-05 IL-06 IL-07 IL-08 IL-10
2
3
4
IL-12
2
3
4
IL-13
0 1 2 3 4
MCP-1 TNFa
0 1 2 3 4
TNFb VEGF
Day1Day2
Day3
3 Days Validation- Standard Curves
25
Automation with Randomization & Normalization
Increased sensitivity
Reduced variability
Rapid development and validation of the new assays
26
COAG™ Chip• Pre-printed multiplex antibody chip (sandwich assay)• Multiplex coagulation-related proteins • High-throughput, low sample volume format• Prognostic biomarker research & risk assessment• Innovation to profile the “coagulome”
27
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Coagulation is Complex
HPCFIX
FVIIFV
Pro
HPC
FIX
FVII
FV
Pro
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
10000.0
Sig
nal (
RFU
)
Capture AntibodyDe
tect
or A
ntib
ody
Antigen Cross Talk ReactivityAg Concentration
10 µg/mL Factor X10 µg/mL Protein C (HPC)1 µg/mL Factor VII100 µg/mL Prothrombin200 µg/mL Factor IX10 µg/mL Factor V
Test: Individual Ag were probed against arrays of capture Abs and then each array was probed individually with cocktails of biotinylated detector antibodies.
Conclusion:Primary antibodies are specific for their respective cognate Ag targets.
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Pro FV
FVIIFIX
HPC
HPC
FIX
FVII
FV
Pro
0
500
1000
1500
2000
2500
3000
Sign
al (R
FU)
Capture Ab
Dete
ctor
Ab
Detector Antibody Cross Talk Reactivity
Test: Physiological cocktails of purified Ag’s were probed against arrays of capture Abs and, then probed with individual specific biotinylated detector antibodies.
Conclusion:
Detector antibodies are specific cognate Ag targets.
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Spot-to-Spot Variability
Sample well of Six-plex Physiological Cocktail
Pro FV FVII FIX FX HPC Control
8 Replicates
Mean St.Dev. %CV PhysiologicalNormal
Prothrombin 8816.5 248.6 2.82 100 µg/mLFactor V 4819.5 189.5 3.93 6.6 µg/mL
Factor VII 10090.3 330.8 3.28 0.5 µg/mLFactor IX 2945.0 66.7 2.26 5.1 µg/mLFactor X 15615.2 731.7 4.69 10 µg/mL
Human Protein C 15615.2 731.7 4.69 3.7 µg/mLPositive Control 48601.8 1335.0 2.75 NA
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Standards
1.0
10.0
100.0
1000.0
10000.0
1.0 10.0 100.0 1000.0 10000.0 100000.0
Antigen Concentration (ng/mL)
Sign
al (R
FU)
Prothrombin Factor VHPC Factor VIIFactor IX Physiological Prothrombin Physiological HPC Physiological Factor VPhysiological Factor VII Physiological Factor IX
Five-plex titration curves
Prothrombin
Factor V
Factor VII
Factor IX
HPC
Standard Dilution Series
8.33 µg/mL 1.39 µg/mL 231 ng/mL 38.6 ng/mL 6.4 ng/mL 1.1 ng/mL
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Normal Human Pooled Plasma versus FIX Immunoaffinity Depleted Plasma
0
2 0
4 0
6 0
8 0
10 0
12 0
14 0
P r o FV FIX HP C% (S
igna
l - B
ackg
roun
d) o
f Nor
mal
Hum
an P
ooPl
asm
a (R
FU)
Normal Human Pool Plasma
Factor IX Depleted Human Plasma
34
Normal Human Pool Plasma vs. HPC Depleted Plasma
0
2 0
4 0
6 0
8 0
10 0
12 0
14 0
16 0
18 0
2 0 0
P ro FV FIX HP C
Normal Human Pool PlasmaHPC Depleted Human Plasma
Normal Human Pool Plasma vs. Prothrombin Depleted Plasma
0
2 0
4 0
6 0
8 0
10 0
12 0
14 0
16 0
18 0
2 0 0
P r o FV FIX HP C
% (S
igna
l - B
ackg
roun
d) o
f Nor
mal
H
uman
Poo
l Pla
sma
(RFU
)
Normal Human Pool Plasma
Prothrombin Depleted Human
Normal Human Pool Plasma vs. Factor V Depleted Plasma
0
2 0
4 0
6 0
8 0
10 0
12 0
14 0
16 0
P r o FV FIX HP C
Normal Human Pool PlasmaFactor V Depleted Human Plasma
Normal Human Pool Plasma vs. FIX Depleted Plasma
0
2 0
4 0
6 0
8 0
10 0
12 0
14 0
P r o FV FIX HP C
Normal Human Pool Plasma
Factor IX Depleted Human Plasma
Normal Human Pooled Plasma versus Immunoaffinitydepleted Plasma
35
CoagulomicsActivated Protein C Resistance (APCR)Annexin VAntithrombin (AT)Antithrombin IIIBeta2 Glycoprotein IC Reactive Protein (CRP)CardiolipinD-DimerFactor IIa-AT ComplexFactor V (Leiden) Mutation AnalysisFactor V (wild type)Factor V HR2 Mutation AnalysisFactor VaFactor VIFactor VIIFactor VIII, FunctionalFactor VIIIcFactor IX
Factor XFactor Xa—AT ComplexFactor XIFactor XIIFactor XIIaFactor XIIIFibrin MonomerFibrino Peptide A (FPA)Fibropeptide BFibrinogenFibronectinHemoclot Thrombin 2Hemoclot Thrombin 8HeparinHirudinHOCl-Oxidized Low Density LipoproteinHomocysteineLipoprotein (a) [Lp(a)]PAI-1 4G/5G ploymorphism
MethylenetetrahydrofolateReductase (MTHFR) MutationPhosphatidylserinePlasminogen Activator Inhibitor-1 (PAI-1) antigen and activityPlasminogen, FunctionalPlatelet Factor 4Protein CProtein SProtein ZProthrombin (factor II) 20210G-A MutationProthrombin (wild-type)ProtimePTTTAFThrombinThrombin-ATIII complexTissue FactorTissue Factor Pathway Inhibitorvon Willebrand Factor (vWF)
36
Single Caputre Ab Arrays• Analog to cDNA and oligo arrays
• Qualitative profiles of protein abundance
• High density profiles
• Proteomic approach to identify smaller panels
37
38
Pandeia™Protein Labeling System
ULS™ labels proteins by forming a coordinative bond:4Sulphur atoms of Methionine and Cysteine4Nitrogen atom in Histidine (pH>5)
R --H2N Pt NH2
H2N
X
SMethionine
SHCysteine
NHN
Histidine
High coverage of human proteome: >98%Less chance of interference with antigen recognition
39
Pandeia™ ProfilingHigh Density Antibody Chips
chipspotted antibodies
mixture of two sera each labeledwith a different hapten-ULS
BIO-ULS FLU-ULS
SA
D647
detection conjugatecocktail
D547
Principle
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Cancer Biomarker Chip
Proposed Protein serum conc. Proposed Protein serum conc.Alpha 2-macroglobulin 131 - 293 mg/dL 28 IL-5 28.45 pg/mLalpha-1-acid glycoprotein 0.5-1.4 mg/mL 29 IL-6 1 - 300 pg/mLalpha-fetoprotein 0.75 - 300 ng/mL 30 IL-6 R 14 - 46 ng/mLb2-Microglobulin 0.01 - 3.2 ug/mL 31 IL-8 25.03 pg/mLCancer antigen 125 1.32 - 531 U/mL 32 Insulin-like growth factor 40-250 ng/mLCancer antigen 15-3 0.24 - 96.8 U/mL 33 Insulin-like growth factor binding protein 3 830 - 3780 ng/mLCancer antigen Sialyl Lewis A (CA 19-9) 0.4 - 159 U/mL 34 Intercellular Cellular Adhesion Molecule-1 211 ng/mLCarcinoembryonic antigen 5.01 - 39.7 ng/mL 35 Interferon gamma < 15 pg/mLCathepsin B 27-126 ng/mL 36 Leutinizing Hormone in serumC-reactive protein 8.22 ug/mL 37 Matrix Metallopeptidase-1 0.9-9 ng/mLEpidermal Growth factor 157.8 pg/mL 38 Matrix Metallopeptidase-2 144 ng/mLEpidermal Growth factor receptor 10 - 3000 ng/mL 39 Matrix Metallopeptidase-3 9.98 ng/mLFAS 4-17 ng/mL 40 Matrix Metallopeptidase-9 <3.5 ng/mLFAS ligand 40-145 pg/mL 41 Monocyte chemoatractant protein-1 200 - 722 pg/mLFibronectin in plasma (serum?) 42 Plasminogen in serumFollicle stimulating hormone in serum 43 Plasminogen activator inhibitor type-1 93.56 ng/mLGranulocyte macrophage colony stimulating factor < 7.8 pg/mL 44 PSA (Free) 0.15 - 46.5 ng/mLHaptoglobin 1-6 mg/mL 45 PSA (Total) 0.12 - 46.5 ug/mLHemoglobin in serum 46 Tissue inhibitor of matrix metalloproteinases-1 206.9 ng/mLHepatocyte growth factor 670 - 2000 pg/mL 47 Tissue inhibitor of matrix metalloproteinases-2 25 - 325 ng/mLIgA 3.61 mg/mL 48 tissue plasminogen activator in serumIgM 1.61 mg/mL 49 Transferrin in serumIL-10 15.53 pg/mL 50 Transforming growth factor-beta 18.3 - 63.4 ng/mLIL-12 p40 1460 pg/mL 51 Vascular Cellular Adhesion Molecule-1 547.8 ng/mLIL-2 10 - 100 pg/mL 52 Vascular endothelial growth factor 354.8 pg/mLIL-3 1050 pg/mL 53 von Willebrand Factor 16.5 ug/mLIL-4 40.12 pg/mL
41
Specificity
Positive controls
AFP
Alpha Feto Protein
0
5000
10000
15000
20000
25000
0.1 1 10 100 1000
Protein concentration (ng/mL)
Bac
kgro
und
subt
ract
ed s
igna
l (R
FU)
Normal Range
42
Cancer Biomarker 1-Color Chip
A2MB2M
CA 19-9
CRPFA
Sfre
e PSA
hapt
oIC
AM-1
IGF
IL-10
IL-3
IL-6
LHMMP-2PAI-1
TGF-
beta
tPA
VEGF
A2M
cat B
fibronectin
ICAM-1
IL-12IL-8
PAI-1transferrin
0
10000
20000
30000
40000
50000
60000
70000A2M AAGAFP B2MCA 125 CA 15-3CA 19-9 cat BCEA CRPEGF EGF-RFAS FAS ligandfibronectin free PSAFSH GM-CSFhapto hemoHGF ICAM-1IFN IgAIGF IGFBP-3IgM IL-10IL-12 IL-2IL-3 IL-4IL-5 IL-6IL-6R IL-8LH MCP-1MMP-1 MMP-2MMP-3 MMP-9PAI-1 plasminogenPSA TGF-betaTIMP-1 TIMP-2tPA transferrinVCAM-1 VEGFvWF
43
Precision
N = 9 labeling reactions using single serum source and added 3 samples to 3 slides.
spot-to-spot well-to-well slide-to-sliden=3 n=3 n=3
% CV % CV % CVSpecification < 20% < 20% < 15%
Average of all protein 8.78% 13.52% 8.75%
44
Breast Cancer vs. Healthy Serum
Comparison of Normal to Diseased Female Serum
0
10000
20000
30000
40000
50000
60000
70000
Bac
kgro
und
subt
ract
ed s
igna
l (R
FU)
Normal Female Serum
Breast Cancer Serum
45
Prostate Cancer vs. Healthy Serum
Comparison of Normal to Diseased Male Serum
0
10000
20000
30000
40000
50000
60000
70000
A2M AFPCA 12
5CA 19
-9
CEA
EGF
FASfib
rone
ctin FSH
hapto HGF
IFN
IGF
IgM
IL-12 IL-3 IL-5
IL-6R LHMMP-1MMP-3
PAI-1 PSATIM
P-1 tPA
VCAM-1 vWF
Bac
kgro
und
subt
ract
ed s
igna
l (R
FU)
Normal Male Serum
Prostate Cancer serum
46
GlycoChipImmobilize Abs on
Nitrocellulose
Oxidize Carbohydrates on Immobilized Abs
Modify with bifunctional linker
(hydrazide-maleimide)
Apend dipeptide(Cys-Gly) to inhibit
lectin binding
47
Lectin Menu
Name of lectin Abbreviation Binds to:1 Aleuria Aurantia Lectin AAL fucose linked (a -1,6) to N-acetylglucosamine or to fucose linked (a -1,3) to N-acetyllactosamine2 Bauhinina Purpurea Lectin BPL galactosyl (b-1,3) N-acetylgalactosamine structures ("T" antigen)3 Lens Culinaris Agglutinin LCA a-linked mannose residues4 Phaseolus vulgaris Leucoagglutinin PHA-L b-1,6 linked mannose5 Ricinus Communis Agglutinin I RCA I oligosaccharides ending in galactose but may also interact with N-acetylgalactosamine6 Sambucus Nigra Lectin SNA Sialic acid7 Wheat Germ Agglutinin WGA N-acetylglucosamine
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Services• Contract Arraying
• Custom Assay Development & Validation
• Aperateur Sample processing services