“Multiplex Immunoassay Development and Validation using ... - Tecan
“Multiplex Immunoassay Development and Validation using ... - Tecan
“Multiplex Immunoassay Development and Validation using ... - Tecan
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
<strong>“Multiplex</strong> <strong>Immunoassay</strong> <strong>Development</strong> <strong>and</strong> <strong>Validation</strong> <strong>using</strong><br />
Ultra-thin Film Nitrocellulose."<br />
Robert Negm, PhD<br />
Vice President, GenTel BioSciences<br />
TECAN SYMPOSIUM 2007
Protein Array Variety<br />
• Quantitative Multiplex <strong>Immunoassay</strong>s<br />
– Cytokine, Metabolic, COAG<br />
– PhosphoArrays<br />
• Single Capture Antibody Arrays<br />
• Antigen Arrays<br />
– Serological Arrays<br />
– Ab Specificity Screening<br />
– Kinase Substrate Profiling/Peptide arrays<br />
• Reverse Western/Lysate arrays<br />
2
PATH<br />
Protein<br />
Microarray<br />
Slide<br />
Conventional<br />
Nitrocellulose<br />
Slide<br />
PATH Performance<br />
Fluorescence Background Comparison<br />
PATH vs Conventional Nitrocellulose Slide<br />
PATH Slide<br />
Conventional<br />
Nitrocellulose<br />
Mean<br />
RFU<br />
398<br />
24998<br />
Std.<br />
Dev.<br />
15<br />
1517<br />
% CV<br />
3.07<br />
6.07<br />
Sample Size<br />
Blank slides scanned at identical parameters. 543 nm excitation<br />
(Cy3). ScanArray® 4000 <strong>and</strong> QuantArray® software. 500<br />
evenly spaced 500 µm circles. PMT = 90, Gain = 75.<br />
500<br />
500<br />
3
Traditional Blood Analyte Measurement<br />
Cytokine S<strong>and</strong>wich <strong>Immunoassay</strong><br />
Antibody Array<br />
Cy3 Labeled Streptavidin<br />
Biotin Labeled Secondary Ab<br />
Antigen (Cytokine)<br />
Primary Ab<br />
4
100000<br />
Signal (RFU)<br />
10000<br />
1000<br />
100<br />
10<br />
5 Logs Dynamic Range<br />
IL-1 Beta<br />
IL-2<br />
IL-6<br />
TNF-Alpha<br />
LOD<br />
0.1 1 10 100 1000 10000 100000<br />
Antigen Concentration (pg/mL)<br />
0.5 pg/mL 5 pg/mL 50 pg/mL 500 pg/mL 5 ng/mL 50 ng/mL<br />
Ambient analyte theory combined with fluoresence.<br />
TNF-Alpha<br />
IL-6<br />
IL-2<br />
IL-1 Beta<br />
5
PATH Performance<br />
Cytokine S<strong>and</strong>wich <strong>Immunoassay</strong><br />
Head-to-head Signal-to-Noise Comparison<br />
6
• Nitrate composition<br />
• Uniform thickness<br />
• Hydrophobicity <strong>and</strong> contact angle<br />
• Protein binding capacity<br />
QA/QC<br />
7
Biomarker Profiling Drivers<br />
• More data with less sample in less time<br />
• Multiple analytes improve clinical sensitivity<br />
<strong>and</strong> specificity in disease diagnostics<br />
• Early detection, benefits of therapy <strong>and</strong><br />
liklihood of disease recurrence<br />
• Surrogate endpoints better than clinical<br />
endpoints<br />
8
Arraying & Imaging<br />
9
PATH HTS<br />
10
• Glass-bottom 3x5 Microplate - design<br />
APiX HTS<br />
• Wells facing up<br />
• Wells facing down<br />
11
Glass-bottom 3x5 Microplate - Assembly<br />
12
Quantitative Multiplex <strong>Immunoassay</strong>s<br />
Detection antibody<br />
Analyte<br />
Capture antibody<br />
13
GenTel Assay <strong>Validation</strong><br />
Methods<br />
– Cross-reactivity<br />
– Dilution Optimization<br />
– St<strong>and</strong>ard Curve <strong>Validation</strong><br />
– Assay Precision<br />
– Dilutional Recovery<br />
14
Eliminating Cross Talk<br />
a) Nonspecific binding between the detector antibodies antigen-capture antibody.<br />
b) Nonspecific binding between the detector antibody with capture antibody.<br />
c) Probing microarrays of capture antibodies with individual antigens.<br />
d) Probing microarrays of capture antibodies with cocktails of antigens.<br />
15
Protein Liquid H<strong>and</strong>ling Process Workflow<br />
Making st<strong>and</strong>ard <strong>and</strong> samples<br />
dilutions<br />
Arranging st<strong>and</strong>ards <strong>and</strong> samples<br />
on a source plate<br />
Transferring st<strong>and</strong>ards <strong>and</strong> samples<br />
from a source plate to chips<br />
Adding detection antibody <strong>and</strong> dye Incubation Washing<br />
16
Image from an Assay Plate<br />
four Chips with 64 Chambers<br />
s<br />
17
Printing format improvement:<br />
reduce variations with scattered<br />
Linear Array<br />
R<strong>and</strong>omized Scattered Array<br />
replicates<br />
18
logSignal<br />
2.5 3.0 3.5 4.0 4.5<br />
Robotic assay <strong>and</strong> normalization protocol<br />
plying normalization (reduced variability)<br />
Analyte = IL-06 (554543) 0.125<br />
No normalized<br />
1.0 1.5 2.0 2.5 3.0 3.5<br />
3.0 3.5 4.0 4.5 5.0<br />
1.0 1.5 2.0 2.5 3.0 3.5<br />
Before normalization After normalization<br />
log10(Conc)<br />
Chip<br />
1 3<br />
2 4<br />
normalization<br />
19
Prob<br />
Prob<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0.0<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0.0<br />
TNFb (551222)<br />
TRAIL (550517)<br />
0.1 0.5 1 5 10 50 100 500 1000<br />
4C:90-min_1<br />
Concentration<br />
0.1 0.5 1 5<br />
4C:90-min_1<br />
10 50<br />
Concentration<br />
100 500 1000 5000<br />
Dilutional Recovery<br />
CV<br />
LLOQ<br />
ULOQ<br />
CV<br />
LLOQ<br />
ULOQ<br />
9 %<br />
2.2<br />
1771.5<br />
9.8 %<br />
10<br />
3765<br />
20
Five (5) Quantitative Multiplex<br />
<strong>Immunoassay</strong>s<br />
21
Cytokine Measurements in Serum, Plasma or Cell Lysates<br />
GM-CSF<br />
IFNγ<br />
IL-1β<br />
IL-2<br />
IL-3<br />
IL-4<br />
IL-5<br />
IL-6<br />
IL-7<br />
IL-8<br />
IL-10<br />
IL-12<br />
IL-13<br />
MCP-1<br />
TNFα<br />
TNFβ<br />
VEGF<br />
22
Human Cytokine Chip Data<br />
23
logSignal<br />
4<br />
3<br />
2<br />
4<br />
3<br />
2<br />
3 Days <strong>Validation</strong>- St<strong>and</strong>ard Curves<br />
IL-13<br />
GM-CSF IFNg<br />
0 1 2 3 4<br />
0 1 2 3 4<br />
MCP-1 TNFa<br />
Dose-Response curves<br />
IL-05 IL-06 IL-07 IL-08 IL-10<br />
IL-01b IL-02<br />
0 1 2 3 4<br />
log10(Conc)<br />
Day1 Day3<br />
Day2<br />
0 1 2 3 4<br />
TNFb VEGF<br />
IL-03 IL-04<br />
0 1 2 3 4<br />
IL-12<br />
4<br />
3<br />
2<br />
24
Automation with R<strong>and</strong>omization & Normalization<br />
Increased sensitivity<br />
Reduced variability<br />
Rapid development <strong>and</strong> validation of the new<br />
assays<br />
25
COAG Chip<br />
• Pre-printed multiplex antibody chip (s<strong>and</strong>wich assay)<br />
• Multiplex coagulation-related proteins<br />
• High-throughput, low sample volume format<br />
• Prognostic biomarker research & risk assessment<br />
• Innovation to profile the “coagulome”<br />
26
Coagulation is Complex<br />
28
Signal (RFU)<br />
10000.0<br />
9000.0<br />
8000.0<br />
7000.0<br />
6000.0<br />
5000.0<br />
4000.0<br />
3000.0<br />
2000.0<br />
1000.0<br />
0.0<br />
HPC<br />
FIX<br />
Capture Antibody<br />
FVII<br />
FV<br />
Pro<br />
Antigen Cross Talk Reactivity<br />
HPC<br />
FIX<br />
FVII<br />
FV<br />
Pro<br />
Detector Antibody<br />
Ag Concentration<br />
10 µg/mL Factor X<br />
10 µg/mL Protein C (HPC)<br />
1 µg/mL Factor VII<br />
100 µg/mL Prothrombin<br />
200 µg/mL Factor IX<br />
10 µg/mL Factor V<br />
Conclusion:<br />
Primary antibodies are<br />
specific for their respective<br />
cognate Ag targets.<br />
Test: Individual Ag were probed against arrays of capture Abs <strong>and</strong> then each array<br />
was probed individually with cocktails of biotinylated detector antibodies.
Signal (RFU)<br />
3000<br />
2500<br />
2000<br />
1500<br />
1000<br />
500<br />
0<br />
Pro<br />
FV<br />
Capture Ab<br />
Detector Antibody Cross Talk Reactivity<br />
FVII<br />
FIX<br />
HPC<br />
HPC<br />
FIX<br />
FVII<br />
FV<br />
Pro<br />
Detector Ab<br />
Conclusion:<br />
Detector antibodies are<br />
specific cognate Ag<br />
targets.<br />
Test: Physiological cocktails of purified Ag’s were probed against arrays of<br />
capture Abs <strong>and</strong>, then probed with individual specific biotinylated detector<br />
antibodies.<br />
30
Spot-to-Spot Variability<br />
Mean St.Dev. %CV<br />
Physiological<br />
Normal<br />
Prothrombin 8816.5 248.6 2.82 100 µg/mL<br />
Factor V 4819.5 189.5 3.93 6.6 µg/mL<br />
Factor VII 10090.3 330.8 3.28 0.5 µg/mL<br />
Factor IX 2945.0 66.7 2.26 5.1 µg/mL<br />
Factor X 15615.2 731.7 4.69 10 µg/mL<br />
Human Protein C 15615.2 731.7 4.69 3.7 µg/mL<br />
Positive Control 48601.8 1335.0 2.75 NA<br />
8 Replicates<br />
Sample well of Six-plex Physiological Cocktail<br />
Pro FV FVII FIX FX HPC Control<br />
31
Prothrombin<br />
Factor V<br />
Factor VII<br />
Factor IX<br />
HPC<br />
Signal (RFU)<br />
10000.0<br />
1000.0<br />
100.0<br />
10.0<br />
St<strong>and</strong>ards<br />
Prothrombin Factor V<br />
HPC Factor VII<br />
Factor IX Physiological Prothrombin<br />
Physiological HPC Physiological Factor V<br />
Physiological Factor VII Physiological Factor IX<br />
1.0<br />
1.0 10.0 100.0 1000.0 10000.0 100000.0<br />
Antigen Concentration (ng/mL)<br />
St<strong>and</strong>ard Dilution Series<br />
Five-plex titration curves<br />
8.33 µg/mL 1.39 µg/mL 231 ng/mL 38.6 ng/mL 6.4 ng/mL 1.1 ng/mL<br />
32
% (Signal - Background) of Normal Human Poo<br />
Plasma (RFU)<br />
14 0<br />
12 0<br />
10 0<br />
80<br />
60<br />
40<br />
20<br />
0<br />
Normal Human Pooled Plasma versus FIX<br />
Immunoaffinity Depleted Plasma<br />
Norm al Human Pool Plasm a<br />
Factor IX Depleted Human Plasma<br />
Pro FV FIX HPC<br />
33
Normal Human Pooled Plasma versus Immunoaffinity<br />
depleted Plasma<br />
14 0<br />
12 0<br />
10 0<br />
16 0<br />
14 0<br />
12 0<br />
10 0<br />
80<br />
60<br />
40<br />
20<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0<br />
Normal Human Pool Plasma vs. FIX Depleted Plasma<br />
Normal Human Pool Plasma<br />
Factor IX Depleted Human Plasma<br />
Pro FV FIX HPC<br />
Normal Human Pool Plasma vs. Factor V Depleted<br />
Plasma<br />
Normal Human Pool Plasma<br />
Factor V Depleted Human Plasma<br />
Pro FV FIX HPC<br />
% (Signal - Background) of Normal<br />
Human Pool Plasma (RFU)<br />
200<br />
18 0<br />
16 0<br />
14 0<br />
12 0<br />
10 0<br />
80<br />
60<br />
40<br />
20<br />
200<br />
18 0<br />
16 0<br />
14 0<br />
12 0<br />
10 0<br />
80<br />
60<br />
40<br />
20<br />
0<br />
0<br />
Normal Human Pool Plasma vs. Prothrombin Depleted<br />
Plasma<br />
Normal Human Pool Plasma<br />
Prothrombin Depleted Human<br />
Pro FV FIX HPC<br />
Normal Human Pool Plasma vs. HPC Depleted<br />
Plasma<br />
Normal Human Pool Plasma<br />
HPC Depleted Human Plasma<br />
Pro FV FIX HPC<br />
34
Activated Protein C Resistance<br />
(APCR)<br />
Annexin V<br />
Antithrombin (AT)<br />
Antithrombin III<br />
Beta2 Glycoprotein I<br />
C Reactive Protein (CRP)<br />
Cardiolipin<br />
D-Dimer<br />
Factor IIa-AT Complex<br />
Factor V (Leiden) Mutation<br />
Analysis<br />
Factor V (wild type)<br />
Factor V HR2 Mutation Analysis<br />
Factor Va<br />
Factor VI<br />
Factor VII<br />
Factor VIII, Functional<br />
Factor VIIIc<br />
Factor IX<br />
Factor X<br />
Factor Xa—AT Complex<br />
Factor XI<br />
Factor XII<br />
Factor XIIa<br />
Factor XIII<br />
Fibrin Monomer<br />
Fibrino Peptide A (FPA)<br />
Fibropeptide B<br />
Fibrinogen<br />
Fibronectin<br />
Hemoclot Thrombin 2<br />
Hemoclot Thrombin 8<br />
Heparin<br />
Hirudin<br />
HOCl-Oxidized Low Density<br />
Lipoprotein<br />
Homocysteine<br />
Lipoprotein (a) [Lp(a)]<br />
PAI-1 4G/5G ploymorphism<br />
Coagulomics<br />
Methylenetetrahydrofolate<br />
Reductase (MTHFR) Mutation<br />
Phosphatidylserine<br />
Plasminogen Activator Inhibitor-1<br />
(PAI-1) antigen <strong>and</strong> activity<br />
Plasminogen, Functional<br />
Platelet Factor 4<br />
Protein C<br />
Protein S<br />
Protein Z<br />
Prothrombin (factor II) 20210G-A<br />
Mutation<br />
Prothrombin (wild-type)<br />
Protime<br />
PTT<br />
TAF<br />
Thrombin<br />
Thrombin-ATIII complex<br />
Tissue Factor<br />
Tissue Factor Pathway Inhibitor<br />
von Willebr<strong>and</strong> Factor (vWF)<br />
35
Single Caputre Ab Arrays<br />
• Analog to cDNA <strong>and</strong> oligo arrays<br />
• Qualitative profiles of protein abundance<br />
• High density profiles<br />
• Proteomic approach to identify smaller<br />
panels<br />
36
P<strong>and</strong>eia<br />
Protein Labeling System<br />
ULS labels proteins by forming a coordinative bond:<br />
4Sulphur atoms of Methionine <strong>and</strong> Cysteine<br />
4Nitrogen atom in Histidine (pH>5)<br />
S<br />
Methionine<br />
H 2N<br />
R --H 2N Pt NH 2<br />
X<br />
SH<br />
Cysteine<br />
N<br />
Histidine<br />
High coverage of human proteome: >98%<br />
Less chance of interference with antigen recognition<br />
NH<br />
38
Principle<br />
detection conjugate<br />
cocktail<br />
mixture of two sera each labeled<br />
with a different hapten-ULS<br />
spotted antibodies<br />
BIO-ULS<br />
P<strong>and</strong>eia Profiling<br />
High Density Antibody Chips<br />
D647<br />
chip<br />
SA<br />
D547<br />
FLU-ULS<br />
39
Cancer Biomarker Chip<br />
Proposed Protein serum conc. Proposed Protein serum conc.<br />
Alpha 2-macroglobulin 131 - 293 mg/dL 28 IL-5 28.45 pg/mL<br />
alpha-1-acid glycoprotein 0.5-1.4 mg/mL 29 IL-6 1 - 300 pg/mL<br />
alpha-fetoprotein 0.75 - 300 ng/mL 30 IL-6 R 14 - 46 ng/mL<br />
b2-Microglobulin 0.01 - 3.2 ug/mL 31 IL-8 25.03 pg/mL<br />
Cancer antigen 125 1.32 - 531 U/mL 32 Insulin-like growth factor 40-250 ng/mL<br />
Cancer antigen 15-3 0.24 - 96.8 U/mL 33 Insulin-like growth factor binding protein 3 830 - 3780 ng/mL<br />
Cancer antigen Sialyl Lewis A (CA 19-9) 0.4 - 159 U/mL 34 Intercellular Cellular Adhesion Molecule-1 211 ng/mL<br />
Carcinoembryonic antigen 5.01 - 39.7 ng/mL 35 Interferon gamma < 15 pg/mL<br />
Cathepsin B 27-126 ng/mL 36 Leutinizing Hormone in serum<br />
C-reactive protein 8.22 ug/mL 37 Matrix Metallopeptidase-1 0.9-9 ng/mL<br />
Epidermal Growth factor 157.8 pg/mL 38 Matrix Metallopeptidase-2 144 ng/mL<br />
Epidermal Growth factor receptor 10 - 3000 ng/mL 39 Matrix Metallopeptidase-3 9.98 ng/mL<br />
FAS 4-17 ng/mL 40 Matrix Metallopeptidase-9
Background subtracted signal (RFU)<br />
25000<br />
20000<br />
15000<br />
10000<br />
5000<br />
Alpha Feto Protein<br />
Normal<br />
Range<br />
0<br />
0.1 1 10 100 1000<br />
Protein concentration (ng/mL)<br />
Specificity<br />
Positive<br />
controls<br />
AFP<br />
41
70000<br />
60000<br />
50000<br />
40000<br />
30000<br />
A2M<br />
B2M<br />
CA 19-9<br />
CRP<br />
FAS<br />
free PSA<br />
hapto<br />
ICAM-1<br />
IGF<br />
IL-10<br />
IL-3<br />
IL-6<br />
LH<br />
MMP-2<br />
PAI-1<br />
20000<br />
10000<br />
0<br />
Cancer Biomarker 1-Color Chip<br />
TGF-beta<br />
tPA<br />
VEGF<br />
A2M AAG<br />
AFP B2M<br />
CA 125 CA 15-3<br />
CA 19-9 cat B<br />
CEA CRP<br />
EGF EGF-R<br />
FAS FAS lig<strong>and</strong><br />
fibronectin free PSA<br />
FSH GM-CSF<br />
hapto hemo<br />
HGF ICAM-1<br />
IFN IgA<br />
IGF IGFBP-3<br />
IgM IL-10<br />
IL-12 IL-2<br />
IL-3 IL-4<br />
IL-5 IL-6<br />
IL-6R IL-8<br />
transferrin LH MCP-1<br />
PAI-1<br />
IL-8<br />
IL-12<br />
ICAM-1<br />
MMP-1<br />
MMP-3<br />
PAI-1<br />
MMP-2<br />
MMP-9<br />
plasminogen<br />
fibronectin PSA TGF-beta<br />
cat B<br />
TIMP-1 TIMP-2<br />
A2M<br />
tPA<br />
VCAM-1<br />
transferrin<br />
VEGF<br />
vWF<br />
42
Precision<br />
spot-to-spot well-to-well slide-to-slide<br />
n=3 n=3 n=3<br />
% CV % CV % CV<br />
Specification < 20% < 20% < 15%<br />
Average of all protein 8.78% 13.52% 8.75%<br />
N = 9 labeling reactions <strong>using</strong> single serum source<br />
<strong>and</strong> added 3 samples to 3 slides.<br />
43
Breast Cancer vs. Healthy Serum<br />
Background subtracted signal (RFU)<br />
70000<br />
60000<br />
50000<br />
40000<br />
30000<br />
20000<br />
10000<br />
0<br />
Comparison of Normal to Diseased Female Serum<br />
Normal Female Serum<br />
Breast Cancer Serum<br />
44
Prostate Cancer vs. Healthy Serum<br />
Background subtracted signal (RFU)<br />
70000<br />
60000<br />
50000<br />
40000<br />
30000<br />
20000<br />
10000<br />
0<br />
Comparison of Normal to Diseased Male Serum<br />
Normal Male Serum<br />
Prostate Cancer serum<br />
A2M<br />
AFP<br />
CA 125<br />
CA 19-9<br />
CEA<br />
EGF<br />
FAS<br />
fibronectin<br />
FSH<br />
hapto<br />
HGF<br />
IFN<br />
IGF<br />
IgM<br />
IL-12<br />
IL-3<br />
IL-5<br />
IL-6R<br />
LH<br />
MMP-1<br />
MMP-3<br />
PAI-1<br />
PSA<br />
TIMP-1<br />
tPA<br />
VCAM-1<br />
vWF<br />
45
Immobilize Abs on<br />
Nitrocellulose<br />
Oxidize<br />
Carbohydrates on<br />
Immobilized Abs<br />
Modify with<br />
bifunctional linker<br />
(hydrazide-maleimide)<br />
Apend dipeptide<br />
(Cys-Gly) to inhibit<br />
lectin binding<br />
GlycoChip<br />
46
Lectin Menu<br />
Name of lectin Abbreviation Binds to:<br />
1 Aleuria Aurantia Lectin AAL fucose linked (a -1,6) to N-acetylglucosamine or to fucose linked (a -1,3) to N-acetyllactosamine<br />
2 Bauhinina Purpurea Lectin BPL galactosyl (b-1,3) N-acetylgalactosamine structures ("T" antigen)<br />
3 Lens Culinaris Agglutinin LCA a-linked mannose residues<br />
4 Phaseolus vulgaris Leucoagglutinin PHA-L b-1,6 linked mannose<br />
5 Ricinus Communis Agglutinin I RCA I oligosaccharides ending in galactose but may also interact with N-acetylgalactosamine<br />
6 Sambucus Nigra Lectin SNA Sialic acid<br />
7 Wheat Germ Agglutinin WGA N-acetylglucosamine<br />
47
• Contract Arraying<br />
• Custom Assay <strong>Development</strong> & <strong>Validation</strong><br />
• Aperateur Sample processing services<br />
Services<br />
48
Change language