Identifying Predictive Cytokine Biomarkers in Asthma Using ... · Program 130.35 / Poster P1261...
1
AAI IMMIUNOLOGY 2019 Technological Innovations I Program 130.35 / Poster P1261 Identifying Predictive Cytokine Biomarkers in Asthma Using the Invitrogen ProQuantum High-Sensitivity Immunoassay Platform David M Bourdon 1 , Helena Ekoff 2 , Anders Sjölander 2 , Takao Fujisawa 3 , Mizuho Nagao 3 , and Magnus Molin 2 1 Thermo Fisher Scientific, USA; 2 Thermo Fisher Scientific, Sweden; 3 Institute for Clinical Research, Mie National Hospital, Japan Introduction Asthma and chronic obstructive pulmonary disease (COPD) affect hundreds of millions of people worldwide and are a significant cause of morbidity and mortality. These heterogenous and complex inflammatory diseases have few good biomarkers for disease etiology, diagnosis, and monitoring of treatment efficacy. Characterization has previously been performed at genomic and proteomic levels, as well as airway histology, lung function, and quality of life metrics. Since inflammation typically involves one or more cytokine cascades, or pathways responsible for triggering and Key Benefits of the ProQuantum High-Sensitivity Immunoassay Platform Sensitivity and broad dynamic range of qPCR A simple, yet powerful platform for target-specific protein quantitation • High-sensitivity – detect low levels of proteins with greater sensitivity than traditional methods like ELISA • Small sample consumption – 2 μL vs 75 μL for triplicate wells with other methods) • Fast, easy workflow – no wash steps, 2 hours from sample to answer • Broad dynamic range – 5 logs minimizes sample dilutions to ensure falling within the range • No proprietary instrument to purchase – runs on any real-time PCR instrument • Includes intuitive cloud-based software – for robust data analysis and statistical group wise comparison Specificity of matched antibody pairs + (c) rob3000 www.fotosearch.se The goal of the following study was to determine whether the Invitrogen ProQuantum High-Sensitivity Immunoassay platform (www.thermofisher.com/proquantum) could prove useful in uncovering biomarker profiles in asthmatic populations. Additional benefits included running the proteomic assay on ubiquitous qPCR instrumentation and the small 2 µL sample requirement. In this study, 168 human serum samples were analyzed (IL-5, IL-6, IL-8, IL-13, IFN-γ and eotaxin-1) across wheezing and asthmatic phenotypes, as well as healthy age- matched controls among both cohorts. Data presented include pairwise analyses that indicate that the ProQuantum high-sensitivity immunoassay has helped uncover predictive biomarkers in samples from wheezing and asthmatic children, that we not previously easily distinguishable. Source: Edwards et al. (2017) Addressing unmet needs in understanding asthma mechanisms, Eur Resp J.49:1602448 maintaining such inflammation, cytokine profiles are critical to advancing our understanding of the disease. However, a powerful immunoassay platform with a simplified workflow that is capable of early cytokine detection and minimum sample consumption is lacking in the asthma / COPD research space. Complementary splint oligo and robust DNA ligase generate qPCR template Antibodies and analyte in a homogeneous mixture Antibodies come into close proximity upon epitope binding Following DNA template ‘melting’ at 95˚C, all proteins (analyte, antibodies, etc.) are denatured and no longer a part of the assay C= cycle number to reach threshold Polymerization Strand displacement Cleavage Merging Powerful and Trusted Genomic and Proteomic Technologies Only need to program qPCR instrument once to create a template for repeated use * * Printable lab instructions (e-notebooks) Standard curve wizard Robust Analytics with Intuitive Cloud-based and Desktop Applications Additional feature: ProQuantum cloud and desktop applications can create GraphPad Prism files for direct integration and convenient creation of publication quality figures Plate setup tool 4PL/5PL analysis with outlier detection Group wise comparison featuring M-stats ProQuantum immunoassays are compatible with any qPCR instrument, and Applied Biosystems instrument users will experience enhanced utility of .EDS files (rapid data import, amplification plots, etc.) Assay Characteristics and Analytics ü Serum spike-and-recovery: 80% - 120% ü Plasma spike-and-recovery: 70% - 130% ü Parallelism and linearity of dilution: >0.9 ü Inter- and intra-assay reproducibility: <15% CV of calculated concentrations (in linear range of assay) ü Analyte cross-reactivity: <10% ü Benchmarking to ELISA ü Protein standard calibration to leading ELISA or NIBSC The ProQuantum Immunoassay Setup Experience in More Detail Prepare reagents Prepare setup plate (on 4˚C cold block) This is a typical example of a setup plate to run the ProQuantum Immunoassay on an entire 96-well plate using 5-µL sample volumes, and a general serial dilution diagram for a standard curve. Reconstitute protein standard Run assay The ProQuantum Immunoassay Workflow Is Rapid, Robust, and Flexible Streamlined Workflow and Intuitive Cloud Application Delivers Rapid Sample-To-Answer Efficiency While Preserving Precious Samples Step 1: antibody-antigen binding • 2* μL 10% serum + 2* μL antibody-conjugates • 1 hr incubation at room temperature Step 2: ligation and qPCR readout • Add 16* μL ligase and qPCR master mix • In-instrument ligation and 40 cycles of qPCR ProQuantum assays are scalable 5 μL volume: one 96-well plate (5+5+40) 2 μL volume: two 96-well plates (2+2+16) 1 μL volume: one 384-well plate (1+1+8) Step 1: Analyte Binding Step 2: Ligation / qPCR 1 2 3 4 Sample binding to plate – 2 hrs W Detection antibody binding – 1.5 hrs W Time (hours) Typical ELISA ProQuantum assays Analyze Results For Additional Information on the ProQuantum High-Sensitivity Immunoassay Platform visit thermofisher.com/ProQuantum For Research Use Only. Not for use in diagnostic procedures. © 2019 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a trademark of Roche Molecular Systems, Inc., used under permission and license. The content provided herein may relate to products that have not been officially released and is subject to change without notice. Results Materials: Six ProQuantum Immunoassays To Detect Circulating Cytokines 16 18 20 22 24 26 28 30 32 34 Concentration, pg/mL C t human IL-5 1,000 100 1 10 0.1 0.01 0.001 0 10,000 16 18 20 22 24 26 28 30 32 Concentration, pg/mL C t human IL-13 1,000 100 1 10 0.1 0.01 0.001 0 10,000 18 20 22 24 26 28 30 32 Concentration, pg/mL C t human IFNγ 1,000 100 1 10 0.1 0.01 0 10,000 18 20 22 24 26 28 30 32 34 Concentration, pg/mL C t human IL-6 1,000 100 1 10 0.1 0.01 0 10,000 16 18 20 22 24 26 28 30 32 34 Concentration, pg/mL C t human IL-8 1,000 100 1 10 0.1 0.01 0.001 0 18 20 22 24 26 28 30 32 Concentration, pg/mL C t human Eotaxin 1,000 100 1 10 0.1 0.01 0 10,000 Example Standard Curves Indicating Assay Range 0 10 20 30 40 0 10 20 30 40 ELISA, pg/mL ProQuantum immunoassay, pg/mL human IL-5 R= 0.8937 0 100 200 300 0 100 200 300 ELISA, pg/mL ProQuantum immunoassay, pg/mL human IL-13 R= 0.8585 0 200 400 600 0 250 500 750 1000 ELISA, pg/mL ProQuantum immunoassay, pg/mL human IFN-γ R= 0.8392 0 50 100 150 200 250 0 50 100 150 200 250 ELISA, pg/mL ProQuantum immunoassay, pg/mL human IL-8 R= 0.9738 0.1 1 10 100 1000 10000 0.01 0.1 1 10 100 1000 10000 Expected concentration, pg/mL ProQuantum immunoassay, pg/mL human IL-6 R= 0.9265 0 20 40 60 80 100 0 20 40 60 80 100 ELISA, pg/mL ProQuantum immunoassay, pg/mL human Eotaxin R= 0.9087 Example Concordance Plots: Same Human Sera Samples Run on ProQuantum Immunoassays and ELISAs ProQuantum Interferon- g Serum conc. (pg/mL) IFN-g wheezing IFN-g healthy 0.01 0.1 1 10 100 1,000 10,000 ProQuantum IL-13 Serum conc. (pg/mL) IL-13 wheezing IL-13 healthy 0.01 0.1 1 10 ProQuantum IL-8 Serum conc. (pg/mL) IL-8 wheezing IL-8 healthy 0.01 0.1 1 10 100 1,000 10,000 ProQuantum Eotaxin Serum conc. (pg/mL) Eotaxin wheezing Eotaxin healthy 1 10 100 ProQuantum IL-5 Serum conc. (pg/mL) IL-5 wheezing IL-5 healthy 0.01 0.1 1 10 100 ProQuantum IL-6 Serum conc. (pg/mL) IL-6 wheezing IL-6 healthy 0.01 0.1 1 10 100 1,000 10,000 • Previously, sensitivity limitations of other immunoassay platforms made it difficult to accurately stratify samples based upon analyte concentration. • In this study 168 serum samples (healthy, wheezing, and asthmatic children) were analyzed across six targets: eotaxin, IL-5, IL-6, IL-8, IL-13, and IFN-!. • ProQuantum High-Sensitivity Immunoassays uncovered marked differences in circulating cytokines levels in human serum samples from both wheezing and asthmatic subjects, versus healthy age-matched controls. ProQuantum IL-5 Serum conc. (pg/mL) IL-5 asthma IL-5 healthy 0.01 0.1 1 10 ProQuantum IL-13 Serum conc. (pg/mL) IL-13 asthma IL-13 healthy 0.001 0.01 0.1 1 Serum conc. (pg/mL) Eotaxin asthma Eotaxin healthy 0.1 1 10 100 ProQuantum Eotaxin Conclusions and Future Directions • The Invitrogen ProQuantum High-Sensitivity Immunoassay platform has been used to detect serum cytokine levels from children afflicted with respiratory disease. • Utilizing the streamlined workflow, rapid sample-to-answer, and without consuming appreciable sample, marked differences have been identified between healthy controls and disease-specific samples. • Future directions include longitudinal analyses on independent sample cohorts to examine early detection profiles, as well as additional analyte targets for study of disease stratification (e.g. eosinophilic asthma versus healthy). Watch the How-To Video
Transcript of Identifying Predictive Cytokine Biomarkers in Asthma Using ... · Program 130.35 / Poster P1261...
AAI IMMIUNOLOGY 2019Technological Innovations IProgram 130.35 / Poster P1261
Identifying Predictive Cytokine Biomarkers in Asthma Using the Invitrogen ProQuantum High-Sensitivity Immunoassay PlatformDavid M Bourdon1, Helena Ekoff2, Anders Sjölander2, Takao Fujisawa3, Mizuho Nagao3, and Magnus Molin2
1Thermo Fisher Scientific, USA; 2Thermo Fisher Scientific, Sweden; 3Institute for Clinical Research, Mie National Hospital, Japan
IntroductionAsthma and chronic obstructive pulmonarydisease (COPD) affect hundreds of millionsof people worldwide and are a significantcause of morbidity and mortality. Theseheterogenous and complex inflammatorydiseases have few good biomarkers fordisease etiology, diagnosis, and monitoringof treatment efficacy. Characterization haspreviously been performed at genomic andproteomic levels, as well as airwayhistology, lung function, and quality of lifemetrics. Since inflammation typicallyinvolves one or more cytokine cascades, orpathways responsible for triggering and
Key Benefits of the ProQuantum High-Sensitivity Immunoassay Platform
Sensitivity and broad dynamic range of qPCR
A simple, yet powerful platform for target-specific protein quantitation
• High-sensitivity – detect low levels of proteins with greater sensitivity than traditional methods like ELISA
• Small sample consumption – 2 μL vs 75 μL for triplicate wells with other methods)
• Fast, easy workflow – no wash steps, 2 hours from sample to answer
• Broad dynamic range – 5 logs minim izes sample dilutions to ensure falling within the range
• No proprietary instrument to purchase – runs on any real-time PCR instrument
• Includes intuitive cloud-based software – for robust data analysis and statistical group wise comparison
Specificity of matched antibody pairs +
(c) rob3000 www.fotosearch.se
The goal of the following study was to determine whether the Invitrogen ProQuantumHigh-Sensitivity Immunoassay platform (www.thermofisher.com/proquantum) couldprove useful in uncovering biomarker profiles in asthmatic populations. Additionalbenefits included running the proteomic assay on ubiquitous qPCR instrumentation and thesmall 2 µL sample requirement. In this study, 168 human serum samples were analyzed (IL-5,IL-6, IL-8, IL-13, IFN-γ and eotaxin-1) across wheezing and asthmatic phenotypes, as well ashealthy age- matched controls among both cohorts. Data presented include pairwise analysesthat indicate that the ProQuantum high-sensitivity immunoassay has helped uncoverpredictive biomarkers in samples from wheezing and asthmatic children, that we notpreviously easily distinguishable.
Source: Edwards et al. (2017) Addressing unmet needs in understanding asthma mechanisms, Eur Resp J.49:1602448
maintaining such inflammation, cytokine profiles are critical to advancing our understanding ofthe disease. However, a powerful immunoassay platform with a simplified workflow thatis capable of early cytokine detection and minimum sample consumption is lacking inthe asthma / COPD research space.
Complementary
splint oligo and
robust DNA ligase
generate qPCR
template
Antibodies and
analyte in a
homogeneous
m ixture
Antibodies come into
close proxim ity upon
epitope binding
Following DNA
template ‘melting’ at
95˚C, all proteins
(analyte, antibodies,
etc.) are denatured
and no longer a part
of the assay
Ct = cycle number to reach threshold
Polymerization
Strand displacement
Cleavage
Merging Powerful and Trusted Genomic and Proteomic Technologies
Only need to program qPCR instrument once to create a template for repeated use
* Note: Sample
dilution can be reduced to 1 µL
+ 9 µL diluent, or any 10-fold
dilution
*
Printable lab instructions (e-notebooks) Standard curve wizard
Robust Analytics with Intuitive Cloud-based and Desktop Applications
Additional feature: ProQuantum cloud and desktop applications can create GraphPad Prism files for direct integration and convenient creation of publication quality figures
Plate setup tool
4PL/5PL analysis with outlier detection
Group wise comparison featuring M-stats
ProQuantum immunoassays are compatible with any qPCR instrument, and Applied Biosystems
instrument users will experience enhanced utility of .EDS files (rapid data import, amplification plots, etc.)
Assay Characteristics and Analyticsü Serum spike-and-recovery: 80% - 120%ü Plasma spike-and-recovery: 70% - 130%ü Parallelism and linearity of dilution: >0.9
ü Inter- and intra-assay reproducibility: <15% CV of calculated concentrations (in linear range of assay)
ü Analyte cross-reactivity: <10% ü Benchmarking to ELISAü Protein standard calibration to
leading ELISA or NIBSC
The ProQuantum Immunoassay Setup Experience in More Detail
Prepare reagentsPrepare setup plate (on 4˚C cold block)
This is a typical example of a setup plate to run the ProQuantum Immunoassay on an entire 96-well plate using 5-µL sample volumes, and a general serial dilution diagram for a standard curve.
Reconstituteprotein
standard
Run assay
The ProQuantum Immunoassay Workflow Is Rapid, Robust, and FlexibleStreamlined Workflow and Intuitive Cloud Application Delivers Rapid Sample-To-Answer Efficiency While Preserving Precious Samples
Step 1: antibody-antigen binding
• 2* μL 10% serum + 2* μL antibody-conjugates
• 1 hr incubation at room temperature
Step 2: ligation and qPCR readout
• Add 16* μL ligase and qPCR master mix
• In-instrument ligation and 40 cycles of qPCR
* ProQuantum assays are scalable5 μL volume: one 96-well plate (5+5+40)2 μL volume: two 96-well plates (2+2+16)1 μL volume: one 384-well plate (1+1+8)
Step 1:Analyte Binding
Step 2:Ligation / qPCR
1 2 3 4
Sample binding to plate – 2 hrs W Detection antibody binding – 1.5hrs W
S u b s tr a te , s to p ,
r e a d
Time (hours)
Typical ELISA
ProQuantumassays
Analyze Results
For Additional Information on the ProQuantum High-Sensitivity Immunoassay Platform visit thermofisher.com/ProQuantum
For Research Use Only. Not for use in diagnostic procedures. © 2019Thermo Fisher Scientific Inc. All rights reserved. All trademarks are theproperty of Thermo Fisher Scientific and its subsidiaries unless otherwisespecified. TaqMan is a trademark of Roche Molecular Systems, Inc., usedunder perm ission and license. The content provided herein may relate toproducts that have not been officially released and is subject to change
without notice.
Results
Materials: Six ProQuantum Immunoassays To Detect Circulating Cytokines
16
18
20
22
24
26
28
30
32
34
Concentration, pg/mL
Ct
human IL-5
1,0001001 100.10.010.0010
10,000
16
18
20
22
24
26
28
30
32
Concentration, pg/mL
Ct
human IL-13
1,0001001 100.10.010.0010
10,000
18
20
22
24
26
28
30
32
Concentration, pg/mL
Ct
human IFNγ
1,0001001 100.10.01010,000
18
20
22
24
26
28
30
32
34
Concentration, pg/mL
Ct
human IL-6
1,0001001 100.10.01010,000
16
18
20
22
24
26
28
30
32
34
Concentration, pg/mL
Ct
human IL-8
1,0001001 100.10.010.0010
18
20
22
24
26
28
30
32
Concentration, pg/mL
Ct
human Eotaxin
1,0001001 100.10.010
10,000
Example Standard Curves Indicating Assay Range
0 10 20 30 400
10
20
30
40
ELISA, pg/mL
Pro
Qua
ntum
imm
unoa
ssay
, pg/
mL human IL-5
R2 = 0.8937
0
100
200
300
0
100
200
300
ELISA, pg/mL
Pro
Qua
ntum
imm
unoa
ssay
, pg/
mL human IL-13
R2 = 0.8585
0
200
400
600
0
250
500
750
1000
ELISA, pg/mL
Pro
Qua
ntum
imm
unoa
ssay
, pg/
mL human IFN-γ
R2 = 0.8392
0 50 100
150
200
250
0
50
100
150
200
250
ELISA, pg/mL
Pro
Qua
ntum
imm
unoa
ssay
, pg/
mL human IL-8
R2 = 0.9738
0.1 1 10 100 1000 100000.01
0.1
1
10
100
1000
10000
Expected concentration, pg/mL
Pro
Qua
ntum
imm
unoa
ssay
, pg/
mL human IL-6
R2 = 0.9265
0 20 40 60 80 100
0
20
40
60
80
100
ELISA, pg/mL
Pro
Qua
ntum
imm
unoa
ssay
, pg/
mL human Eotaxin
R2 = 0.9087
Example Concordance Plots: Same Human Sera Samples Run on ProQuantum Immunoassays and ELISAs
Eosinophil-DerivedNeurotoxin (EDN)
Seru
m c
onc.
(ng/
mL)
EDN whe
ezing
EDN health
y1
10
100
1,000
ProQuantum IL-5
Seru
m c
onc.
(pg/
mL)
IL-5 whee
zing
IL-5 hea
lthy
0.01
0.1
1
10
100
ProQuantum IL-13
Seru
m c
onc.
(pg/
mL)
IL-13 w
heezin
g
IL-13 hea
lthy
0.01
0.1
1
10
ProQuantum Eotaxin
Seru
m c
onc.
(pg/
mL)
Eotaxin w
heezin
g
Eotaxin hea
lthy
1
10
100
ProQuantum IL-6
Seru
m c
onc.
(pg/
mL)
IL-6 w
heezin
g
IL-6 h
ealth
y0.01
0.1
1
10
100
1,000
10,000
ProQuantum IL-8
Seru
m c
onc.
(pg/
mL)
IL-8 w
heezin
g
IL-8 h
ealth
y0.01
0.1
1
10
100
1,000
10,000ProQuantum Interferon- g
Seru
m c
onc.
(pg/
mL)
IFN-g
wheezin
g
IFN-g
health
y0.01
0.1
1
10
100
1,000
10,000Eosinophil-DerivedNeurotoxin (EDN)
Seru
m c
onc.
(ng/
mL)
EDN whe
ezing
EDN health
y1
10
100
1,000
ProQuantum IL-5
Seru
m c
onc.
(pg/
mL)
IL-5 whee
zing
IL-5 hea
lthy
0.01
0.1
1
10
100
ProQuantum IL-13
Seru
m c
onc.
(pg/
mL)
IL-13 w
heezin
g
IL-13 hea
lthy
0.01
0.1
1
10
ProQuantum Eotaxin
Seru
m c
onc.
(pg/
mL)
Eotaxin w
heezin
g
Eotaxin hea
lthy
1
10
100
ProQuantum IL-6
Seru
m c
onc.
(pg/
mL)
IL-6 w
heezin
g
IL-6 h
ealth
y0.01
0.1
1
10
100
1,000
10,000
ProQuantum IL-8Se
rum
con
c. (p
g/m
L)
IL-8 w
heezin
g
IL-8 h
ealth
y0.01
0.1
1
10
100
1,000
10,000ProQuantum Interferon- g
Seru
m c
onc.
(pg/
mL)
IFN-g
wheezin
g
IFN-g
health
y0.01
0.1
1
10
100
1,000
10,000
Eosinophil-DerivedNeurotoxin (EDN)
Seru
m c
onc.
(ng/
mL)
EDN whe
ezing
EDN health
y1
10
100
1,000
ProQuantum IL-5
Seru
m c
onc.
(pg/
mL)
IL-5 whee
zing
IL-5 hea
lthy
0.01
0.1
1
10
100
ProQuantum IL-13
Seru
m c
onc.
(pg/
mL)
IL-13 w
heezin
g
IL-13 hea
lthy
0.01
0.1
1
10
ProQuantum Eotaxin
Seru
m c
onc.
(pg/
mL)
Eotaxin w
heezin
g
Eotaxin hea
lthy
1
10
100
ProQuantum IL-6
Seru
m c
onc.
(pg/
mL)
IL-6 w
heezin
g
IL-6 h
ealth
y0.01
0.1
1
10
100
1,000
10,000
ProQuantum IL-8
Seru
m c
onc.
(pg/
mL)
IL-8 w
heezin
g
IL-8 h
ealth
y0.01
0.1
1
10
100
1,000
10,000ProQuantum Interferon- g
Seru
m c
onc.
(pg/
mL)
IFN-g
wheezin
g
IFN-g
health
y0.01
0.1
1
10
100
1,000
10,000
Eosinophil-DerivedNeurotoxin (EDN)
Seru
m c
onc.
(ng/
mL)
EDN whe
ezing
EDN health
y1
10
100
1,000
ProQuantum IL-5
Seru
m c
onc.
(pg/
mL)
IL-5 whee
zing
IL-5 hea
lthy
0.01
0.1
1
10
100
ProQuantum IL-13
Seru
m c
onc.
(pg/
mL)
IL-13 w
heezin
g
IL-13 hea
lthy
0.01
0.1
1
10
ProQuantum Eotaxin
Seru
m c
onc.
(pg/
mL)
Eotaxin w
heezin
g
Eotaxin hea
lthy
1
10
100
ProQuantum IL-6
Seru
m c
onc.
(pg/
mL)
IL-6 w
heezin
g
IL-6 h
ealth
y0.01
0.1
1
10
100
1,000
10,000
ProQuantum IL-8Se
rum
con
c. (p
g/m
L)
IL-8 w
heezin
g
IL-8 h
ealth
y0.01
0.1
1
10
100
1,000
10,000ProQuantum Interferon- g
Seru
m c
onc.
(pg/
mL)
IFN-g
wheezin
g
IFN-g
health
y0.01
0.1
1
10
100
1,000
10,000
Eosinophil-DerivedNeurotoxin (EDN)
Seru
m c
onc.
(ng/
mL)
EDN whe
ezing
EDN health
y1
10
100
1,000
ProQuantum IL-5
Seru
m c
onc.
(pg/
mL)
IL-5 whee
zing
IL-5 hea
lthy
0.01
0.1
1
10
100
ProQuantum IL-13
Seru
m c
onc.
(pg/
mL)
IL-13 w
heezin
g
IL-13 hea
lthy
0.01
0.1
1
10
ProQuantum Eotaxin
Seru
m c
onc.
(pg/
mL)
Eotaxin w
heezin
g
Eotaxin hea
lthy
1
10
100
ProQuantum IL-6
Seru
m c
onc.
(pg/
mL)
IL-6 w
heezin
g
IL-6 h
ealth
y0.01
0.1
1
10
100
1,000
10,000
ProQuantum IL-8
Seru
m c
onc.
(pg/
mL)
IL-8 w
heezin
g
IL-8 h
ealth
y0.01
0.1
1
10
100
1,000
10,000ProQuantum Interferon- g
Seru
m c
onc.
(pg/
mL)
IFN-g
wheezin
g
IFN-g
health
y0.01
0.1
1
10
100
1,000
10,000
Eosinophil-DerivedNeurotoxin (EDN)
Seru
m c
onc.
(ng/
mL)
EDN whe
ezing
EDN health
y1
10
100
1,000
ProQuantum IL-5
Seru
m c
onc.
(pg/
mL)
IL-5 whee
zing
IL-5 hea
lthy
0.01
0.1
1
10
100
ProQuantum IL-13
Seru
m c
onc.
(pg/
mL)
IL-13 w
heezin
g
IL-13 hea
lthy
0.01
0.1
1
10
ProQuantum Eotaxin
Seru
m c
onc.
(pg/
mL)
Eotaxin w
heezin
g
Eotaxin hea
lthy
1
10
100
ProQuantum IL-6
Seru
m c
onc.
(pg/
mL)
IL-6 w
heezin
g
IL-6 h
ealth
y0.01
0.1
1
10
100
1,000
10,000
ProQuantum IL-8
Seru
m c
onc.
(pg/
mL)
IL-8 w
heezin
g
IL-8 h
ealth
y0.01
0.1
1
10
100
1,000
10,000ProQuantum Interferon- g
Seru
m c
onc.
(pg/
mL)
IFN-g
wheezin
g
IFN-g
health
y0.01
0.1
1
10
100
1,000
10,000
• Previously, sensitivity limitations of other immunoassay platforms made it difficult to accurately stratify samples based upon analyte concentration.
• In this study 168 serum samples (healthy, wheezing, and asthmatic children) were analyzed across six targets: eotaxin, IL-5, IL-6, IL-8, IL-13, and IFN-!.
• ProQuantum High-Sensitivity Immunoassays uncovered marked differences in circulating cytokines levels in human serum samples from both wheezing and asthmatic subjects, versus healthy age-matched controls.
EDN
Seru
m c
onc.
(ng/
mL)
EDN asthma
EDN health
y1
10
100
1,000
ProQuantum IL-5
Seru
m c
onc.
(pg/
mL)
IL-5 a
sthma
IL-5 h
ealth
y0.01
0.1
1
10ProQuantum IL-13
Seru
m c
onc.
(pg/
mL)
IL-13 as
thma
IL-13 hea
lthy
0.001
0.01
0.1
1
ProQuantum eotaxin
Seru
m c
onc.
(pg/
mL)
Eotaxin
asthm
a
Eotaxin
healt
hy0.1
1
10
100
EDN
Seru
m c
onc.
(ng/
mL)
EDN asthma
EDN health
y1
10
100
1,000
ProQuantum IL-5
Seru
m c
onc.
(pg/
mL)
IL-5 a
sthma
IL-5 h
ealth
y0.01
0.1
1
10ProQuantum IL-13
Seru
m c
onc.
(pg/
mL)
IL-13 as
thma
IL-13 hea
lthy
0.001
0.01
0.1
1
ProQuantum eotaxin
Seru
m c
onc.
(pg/
mL)
Eotaxin
asthm
a
Eotaxin
healt
hy0.1
1
10
100
EDN
Seru
m c
onc.
(ng/
mL)
EDN asthma
EDN health
y1
10
100
1,000
ProQuantum IL-5
Seru
m c
onc.
(pg/
mL)
IL-5 a
sthma
IL-5 h
ealth
y0.01
0.1
1
10ProQuantum IL-13
Seru
m c
onc.
(pg/
mL)
IL-13 as
thma
IL-13 hea
lthy
0.001
0.01
0.1
1
ProQuantum eotaxin
Seru
m c
onc.
(pg/
mL)
Eotaxin
asthm
a
Eotaxin
healt
hy0.1
1
10
100
ProQuantum Eotaxin
Conclusions and Future Directions• The Invitrogen ProQuantum High-Sensitivity Immunoassay platform has been used to
detect serum cytokine levels from children afflicted with respiratory disease.
• Utilizing the streamlined workflow, rapid sample-to-answer, and without consuming appreciable sample, marked differences have been identified between healthy controls and disease-specific samples.
• Future directions include longitudinal analyses on independent sample cohorts to examine early detection profiles, as well as additional analyte targets for study of disease stratification (e.g. eosinophilic asthma versus healthy).
Watch the How-To Video
