Multiplexing Makes Scientific Sense “ Luminex xMAP Technology Multiplexing Makes Scientific Sense...

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!