“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

4

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.

6

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

10

PATH™ HTS

11

APiX™ HTS• Glass-bottom 3x5 Microplate - design

• Wells facing up

• Wells facing down

12

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

20

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

21

Five (5) Quantitative Multiplex Immunoassays

22

Cytokine Measurements in Serum, Plasma or Cell Lysates

GM-CSFIFNγIL-1βIL-2IL-3IL-4IL-5IL-6IL-7IL-8

IL-10IL-12IL-13

MCP-1TNFαTNFβVEGF

23

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

28

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.

30

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.

31

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

32

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

33

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

40

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

48

Services• Contract Arraying

• Custom Assay Development & Validation

• Aperateur Sample processing services