multiplexing makes scientific sense “ luminex xmap technology multiplexing makes scientific sense...
TRANSCRIPT
Luminex xMAP Technology
Multiplexing Makes Scientific SenseMultiplexing Makes Scientific Sense “ “What you don´t know won´t hurt you.......or will it?”
Alan Tunnicliffe
xMAP provides data quickly & accurately
- xMAP Technology – The Principle
- Luminex Platforms – Brief Overview
Applications – Arguments are won with Data!
- Cytokines – Screening & Profiling
- Cancer Prediction and staging
- Human Metabolism Panels
- Cell Signaling – Next Generation of Analysis
Contents
The History Bit
Ignorance more frequently begets confidence than does knowledge: it is those who know little, not those who know much, who so positively assert that this or that problem will never be solved by science. (Charles Darwin, Introduction to The Descent of Man, 1871)
5 years on HMS Beagle- data collection
The Geography Bit.....
Classic Immunoassays such as
ELISA, Western blot and RIAs
are very accurate and precise -
BUT they tell you very little
about the surroundings.
Back to Darwin
“”””But I look with confidence to the future to young and rising naturalists, who will be able to view both sides of the question with impartiality”””””.
It took Darwin over 20 years to publish-
Nowadays that would be a time frame few Modern day scientists can accept...........
Therefore, techniques which generate a mass of data must also have a means to assimilate, condense and analyse down to meaningful and manageable information in a reasonable time
Why is one piece of data misleading?
. because there is variation in every population
The challenge of the modern scientist is to devise a model that accounts for a significant proportion of outcomes.
One must take into account factors such as`:
Age
Weight
Sex
Geography
Culture etc
Therefore a model should have a wealth of data in support that can allow for these factors and still be predictive.
Thus there is a need for more data and not less- and then processing the data is the key
The classical data producers.
Situation Problem What does that
mean
How can Multiplexing address the
problem
Western blot One protein at a time; qualitative
Time, reagents and sample wasted running multiple assay
Able to screen multiple proteins; quantitative
ELISAOne protein at a time
Time, reagents and sample wasted running multiple assay
Able to screen multiple proteins: Total and activated at same time
Traditional cell signaling assays
Qualitative; ~10 analytes max; may require 3+ wells for analysis
Often want/need quantitation; time wasted running multiple assays
Quantitative; large panels possible; analyze multiple phospho-sites and total in the same well
Challenges You Face
Limited Resources
Staffing Equipment Supplies
Limited sample
Small volumes available Restricted number of patients Reproducibility of data
Limited Time
Need to generate data Writing and publishing papers Writing grants
Need to do more with less!
ELISA: 1 cytokine/well
SECRETED PROTEINS
CELL SIGNALING PROTEINS
WESTERN: 1 protein/probe
1 protein/ sample!!
Data correlation to Luminex
When comparing Luminex to traditional technologies, ALL methods must use the same antibodies, standards, samples, and buffers to be equally comparable.
Leptin Levels in human Serum Samples
y = 0.5789x - 68.771
R2 = 0.919
0
500
1000
1500
2000
0 500 1000 1500 2000 2500 3000 3500
ELISA (pM)
Linc
ople
x A
dipo
kine
B
(pM
)
Leptin Levels in Human Serum Samples
y = 0.7868x + 36.165
R2 = 0.9425
0500
10001500
2000250030003500
0 500 1000 1500 2000 2500 3000 3500 4000
RIA (pM)
Linc
ople
x (p
M)
Multiplexing – overcoming the challenge
To obtain more data points per sample, it must be attractive to:
- measure several analytes at the same time
- use a small amount of sample
- measure all analytes under the same conditions (no additional storage, freeze-thaw issues)
- and if all assays are developed by the same manufacturer - gives consistency, correlation and confidence
=> Multiplexing makes scientific and economical sense
The Multiplex xMAP® Difference
Number of plates required
Total time to result
Results per plate
Total sample used per panel
Internal controls possible?
Dynamic Range
Lower limit of detection
10 proteins10 proteins40 samples40 samplesExample: Human Cytokine kit
ELISAELISA
10 hours10 hours
8080
4 ml4 ml
NONO
10-2500 pg/ml10-2500 pg/ml
~1 pg/ml~1 pg/ml
xxMAPMAP®® TechnologyTechnology
3 hours3 hours
800800
YESYES
50 50 ll
1-10,000 pg/ml1-10,000 pg/ml
~1 pg/ml~1 pg/ml
Economic reasons for Multiplexing
3 analytes
Substantial cost savings due to:> Less sample to use
> Lower labor costs
13
Increasing Rate of MILLIPLEX Publications
0
50
100
150
200
250
300
350
2004 2005 2006 2007 2008 2009 2010
Year of Publication
Num
ber
of
Pub
licat
ions
*
*Data was compiled on 17.04.11 through searching PubMed for keyword Luminex OR Lincoplex OR MILLIPLEX OR Beadlyte
Data correlation to Luminex
When comparing Luminex to traditional technologies, ALL methods must use the same antibodies, standards, samples, and buffers to be equally comparable.
Leptin Levels in human Serum Samples
y = 0.5789x - 68.771
R2 = 0.919
0
500
1000
1500
2000
0 500 1000 1500 2000 2500 3000 3500
ELISA (pM)
Linc
ople
x A
dipo
kine
B
(pM
)
Leptin Levels in Human Serum Samples
y = 0.7868x + 36.165
R2 = 0.9425
0500
10001500
2000250030003500
0 500 1000 1500 2000 2500 3000 3500 4000
RIA (pM)
Linc
ople
x (p
M)
Luminex xMAP: The Microspheres
magnetite
magnetic
Non-magnetic
> Polystyrene microsphere• 5.6 micron MicroPlex• 6.5 micron MagPlex
> Carboxylated surface
> Small bead size> Suspension in liquid> Fast kinetics> High surface-to-volume ratio
R
Luminex Coupling Proteins to Beads
Conventional sandwich immunoassay technology or competitive assays
Capture antibody
Luminex-Bead
Y YY YAnalyte
Biotinylated detection antibody
SA-PE
Y Y
Y
Y
Carboxy Beads => covalent coupling of antibodies, proteins... Carboxy Beads => covalent coupling of antibodies, proteins...
Microplex: 1 x 10E8 binding sites/ bead
Two fluorescent dyes, one red and one infrared are mixed in precise proportions in organic solvents.
When the polystyrene microspheres are added they swell in the organic solvent. This allows the precise proportion of red and infrared dyes to diffuse into the interior of the microspheres.
MicroPlex® Beads
Luminex then moves the microspheres back into aqueous solution, which shrinks the microspheres and traps this precise proportion of red and infrared dye in the interior.
MicroPlex® Beads
5.6 micron (non magnetic) or 6.5 micron (magnetic) beads
Up to 100 (500) different beads/well
- Bead color = spectral address
- A combination of red and infrared (& orange red) dyes distinguishes one bead set from another
Small bead size allows:
Liquid suspension assay with
- a high surface-to-volume-ratio
- fast kinetics (liquid-phase behaviors)
xMAP® Technology - Multi-Analyte Profiling
Luminex Assay Priniciple
Wash 2x
25µl Detection Ab
Incubate (shake), no Wash
Prewet 96 well plate with 200µl Assay Buffer
Shake, Wash
10 to 25µl* Std/ Sample + 25µl* Matrix/Buffer + 25µl* Beads
Incubate (shake)
25µl Streptavidin-Phycoerythrin
Incubate (shake)Wash 2x
150µl Sheath Fluid
Read Plate on Luminex Machine
* Depends on panel!!
General Assay Protocol (wash)
Instrumentation Portfolio
Non-Mag & Mag BeadsMag Beads only!!
Identify bead region based on internal dye concentrations
Quantify binding events
Interrogate bead with red laser (635 nm)
Interrogate reporter dye (PE) with green laser (532 nm)
Sheath FluidHydrodynamic Focusing of Sample
Flow Cytometry-based analysis 10 sec dwell time
=> red laser for bead classification => allows multiplexing=> green laser for assay result => allows quantification
LX200/FM3D: Detection - The Lasers
Interrogation of the Microspheres:
MAGPIX - Fluorescence Detection
Sample aspirated into the instrument
Beads enter magnetic chamber & form monolayer
=> for imaging Red LED excites internal bead dyes
- Determines which analyte is being measured
=> Green LED excites detection (PE) Fluorophores
- Measures expression (correlates with conc.)
CL1 Filter
CL2 Filter
RP1 Filter
LED Illumination Capture image with CCD camera and appropriate filter
Image Analysis
Two classification images will identify which bead set each individual bead is from – identifies assay
Reporter image quantifies assay – is your target analyte present and at what level?
Bead Classification
Assay Reporting
MAGPIX: Classification & Reporter Images
MAGPIX: 50 Region Bead MapC
las
sifi
cati
on
2
Classification 1
Accuracy/Precision Efficiency Flexibility
What factors should you think about when choosing a technology?
xMAP Benefits
True representative quality control
Sensitive assays
Internal controls
More data in less time by multiplexing
Reduced sample, labor, and costs
Multiformat platform ideal for core labs
Multi-format – DNA, immuno, enzyme
Kits or custom assays
Expand or reduce assay panels easily
Is a technology the answers to everyone’s needs?
validation
Narrowing D
own
Dis
cove
ry
# o
f an
alyt
es
100’s -
10’s -
10’s 1000’s
# of samples
Bead based multiplexing
100’s 10,000’s
ELISAs/RIAs
xMAP Technology is Flexible
xMAP provides data quickly & accurately
- xMAP Technology – The Principle
- Luminex Platforms – Brief Overview
Applications – Arguments are won with Data!
- Cytokines – Screening & Profiling
- Human Metabolism Panels
- Cell Signaling – Next Generation of Analysis
Contents
Circulating Biomarkers(Metabolic Diseases, Inflammation/ Immunology, Oncology, Neuroscience, Toxicity)
Intracellular proteins(Cell Signaling Pathways)
Nuclear
Blood Vessel
DNA
Transcription factorsCell functions Cell functions
Extracellular
Intracellular
GPCRCytokine receptor Ca2+
Biomarker Panel in general
Cytokine Interactions
Cytokine Screening in Normal and Septic Serum
1
2
3
4
5
6
7
8
Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
GM-CSF
IFN
IL-10
IL-12p70
IL-6
IL-8
Cerebral Spinal FluidCerebral Spinal Fluid
Patient 1 Patient 2 Patient 3 Patient 4 Patient 5
200
400
600
800
1000
1200
1400
1600
1800
pg
/ml
pg
/ml
Lumbar punctures were performed on patients with encephalitis and meningitis suspected of Lumbar punctures were performed on patients with encephalitis and meningitis suspected of West Nile Virus infection. Aliquots were frozen and later 50West Nile Virus infection. Aliquots were frozen and later 50l was analyzed for 10 cytokinesl was analyzed for 10 cytokines
Cytokine/Chemokines in CSF: Experimental Data
Ovarian Cancer
Epithelial ovarian Carcinoma is the leading cause of death from gynecologic cancers.
Presents with advanced stage disease
Major Reason-lack of effective screening
Sensitive Method that can distinguish:
Normal vs. ovarian cancer patients
Stage I and II of disease
Proteomics
Mass spectrometry
2D-Gels
Genomics
Micro arrays
169 Proteins Tested from 46 Patients (28 healthy and 18 cancer) (micro arrays)
35 Leads-Proteins Filtered from Group I 112 recurrent ovarian cancer patients(micro arrays)
10 statistically significant proteins (micro arrays)
6 Proteins from Group III Tested with ELISA on 50 Patient Samples
4 Proteins selected for a blind study (206) and analyzed as clusters.Statistical Analysis Confirmed Significant Differentiation between Normal (106) and Cancer Patients (100)
I
II
III
IV
V
STAGE Screening Process
Ovarian Cancer Panel DevelopmentScreening Method for Early Detection Proteins
Different Sample Population
Different Methods of Evaluation
Blind Study
Multiplex
Correlative data with ELISA
Ovarian Cancer Panel (the initial panel)Ovarian Cancer Panel (the initial panel)
Mor et al Proc Natl Acad Sci U S A. 2005 May 24;102(21):7677-82.
Detection %correctPhase I/II 23/24 96%Phase III/IV 102/108 95%Normals 110/114 96%
Multiplexing Results
In order to develop a screening test it is necessary to achieve a specificity of 99.6%
Use of additional two markers: MIF and CA125
Final analysis
Sensitivity : 97.4 %
Specificity : 99.77%
PPV: 99.35%
NPV: 98.7%
Trends in Obesity
The Role of Adipose Tissue
Hormonescytokines,
growth factors, agonists, antagonists
Environment
GeneticsLipolysis
Lipogenesis
Proliferation Differentiation
Apoptosis
TNF
Leptin
Adiponectin
Resistin
AGT
PAI-1
LPL
HSL
Insulin
TZDs
IL-6
Measuring these changes?
Childhood Obesity
Clinical features of obese children and adolescents
60% 60%
53% 59%
Family history of diabetesof obesity
102.8±1.9112.9±2.7 Waist, cm
3.6±0.063.8±0.08 SDS-BMI
36.1±0.637.2±0.8BMI, kg/m²
14.5 (8-18)14.5 (9-18)Age, mean (range)
Girls Boys
Inflammatory markers in obese children with and without the metabolic syndrome
0.00
2.00
4.00
6.00
8.00
Obese IR High TGLow HDL
MS
Me
an
TN
Fα
ng
/mL
p<0.01p<0.01
Me
an
CR
P n
g/m
L
Obese IR High TGLow HDL
MS
0.00
5,000.00
10,000.00
15,000.00
20,000.00 p<0.01
Obese IR High TGLow HDL
MS0.00
10,000.00
20,000.00
30,000.00
40,000.00M
ea
n P
AI-
1 p
g/m
L
p<0.05
1.00 2.00 3.00 4.000.00
10,000.00
20,000.00
30,000.00
40,000.00
50,000.00
Me
an
fib
rin
og
en
ng
/mL p<0.05
Sex and age adjusted odds ratio of having metabolic syndrome
0
2
4
6
8
10
12
adiponectin
≤ median 9 µg/ml
uric acid PAI-1 IL-18 CRP fibrinogen
≥ median
(95% CI 1.12-7.49)
P<0.05
2.9
Only adiponectin independently
associated with risk of Metabolic
syndrome(p<0.001)
1.1
NS
2.7
NS
1.8
NS
(95% CI 2.13-14.27)P<0.0001
5.5
(95% CI 2.97-38.99)
P<0.0001
10.7
Cell Signaling Panels- Intracellular Biomarkers
Cell signaling is part of a complex system of communication that dictates basic activities in the cell and coordinates cellular functions.
Errors in cellular information processing are responsible for diseases such as cancer, autoimmunity and diabetes.
A greater understanding of cell signaling has and will continue to identify targets for therapeutic intervention.
The ability to detect PTMs is critical to the understanding of cellular communication and treatment of many disease states.
Citations in PubMed and Number of Modification Sites
Cell Signaling: A Workhorse for Basic Research and Drug Discovery
Reversible phosphorylation is the most common regulator of cellular events. Hence the prominence of kinases in drug discovery research.
Approximately 30% of cellular proteins are regulated by reversible phosphorylation
25,000 Proteins in human proteome
7,500 Proteins may be phosphorylated
Multiple sites (range 1 to 10)
Potential sites 7,500 to 75,000
Sites in public databases (Sept. ’09) 53,262
Generic 4G10 does not report which tyrosine has been phosphorylated.
Therefore, specific monoclonal antibodies against each site are required to fully understand cellular data.
Modification SitesTyrosine: 6 sitesSerine: 3 sites
Lymphocyte-specific protein-tyrosine kinase Activation: Y394Inhibition: Y192Phosphorylation: Y192Regulates Molecular Association: S59, Y192
LCK – Multiple Phosphorylation Sites that Influence Different Cellular Processes
11plex AKT/mTOR-Panel
# 48-611MAG
Ser473
Ser21
Ser9
Tyr1135/1136
Tyr1162/1163
Ser312
Ser2448
Thr412
Ser380
Ser235/236
Ser939
# 48-612MAG
Akt
GSK3a
GSK3b
IGF1R
IR
IRS1
mTOR
P70 S6 Kinase
PTEN
RPS6
TSC2
Panel Overview: Quarterly Product Update
http://www.millipore.com/bmia/flx4/biomarkers-immunoassays-homepage
Summary
The Luminex technology has a number of important advantages over traditional ELISA or Western blot analysis:
Better Results - Quantitative analysis of multiple proteins from a single sample in the same well.
Save Sample – TheAssays typically require max 25µl of sample/ well
Save Time - Parallel analysis of proteins from the same sample means a faster time to data.
Save Money - Combining assays not only saves time, multiplex assays typically cost a fraction of the price of comparable number of ELISAs.
Arguments are won with data!
Thank You!