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Next generation multiplexed microarray

protein screening using Grace Bio-

Labs™ ArrayCAM™ Proteomic

Profiling System

Protein Profiling – Future of Disease Detection

► Profiling sets of multiple protein markers and signaling

pathways will become the standard for future methods of accurate

disease diagnosis and monitoring therapeutic responses.

- Economy of high

throughput multiplex

platforms

- Reproducible, sensitive,

specific results

- Reliable and confident

assay controls

- Easy to use

- Early/accurate

detection

- Enhanced

therapeutic

monitoring

- Increased

biomarker

throughput

- Improved

survivability/man

ageability

- Reduced

healthcare and

R&D costs

Grace Bio-Labs ArrayCAM™ Proteomic Profiling System

Grace Bio-Labs Profiling

System Optimal sensitivity/specificity

Robust reproducibility

Confident assay controls

Unlimited multiplexingOptimized Reagents/Protocols

• Validated easy to use protocols

• Complete, familiar, easy methods

ArrayCAM Imager

• Fast image acquisition

• Benchtop or automated HTS

• Optimized wavelength fluorophores

• Automated image quantitation

ONCYTE Porous Nitrocellulose Film

• Highest protein binding capacity

• Retained tertiary structure

• Unlimited multiplex density

Multi-Color Quantum Nanocrystal

Fluorescence Detection

• Highest signal to noise

• Archivable signal stability

• Unique and robust assay controls

Anti-Nuclear Antibody (ANA) Disease Subset Serology Screening

► Autoimmune diseases result from a dysfunction of the

immune system in which an abnormal immune response

develops to the body’s own organs, tissues, and cells

► ANA Detection Methodology –

-Autoantibodies to dsDNA and histones

– screening

-Autoantibodies to extractable nuclear antigens (ENA)

– complex subset screening, multiple markers

ANA Testing Inadequacies

► Current standard methods of ANA testing present numerous

inadequacies

Platform Method Drawback

Immunoflourescence- Gold standard,

- IF with HepG2 Cell Preps

- Low Throughput

- Subjective results

- Poor specificity

- False positives

EIA/ELISA- Gaining adoption

- Microplate immunoassays

- Poor sensitivity

- False negatives

Multiplexed Flow

Cytometry

- Initial adoption

- Bead immunoassays

- False positives via complexing

- Poor dsDNA results

► ArrayCAM Proteome Profiling System ANA 6-Plex Serology

Assay demonstrates a complete solution to this example of disease

screening via protein profiling

ANA 6-Plex Assay Details► Immobilized Antigen Markers – SM, SM/RNP, SSA, SSB, JO-1, dsDNA

► Array Controls

- BSA – non-specific binding control

- Human IgG Titration – calibration controls

- Anti-Human IgG – absolute sample control

- HepB antigen – absolute sample control

- Printing buffer – negative controls

► Test samples- acquired from three sources

• Immuno Concepts, Inc.• Asterand, USA

• RDL Reference Laboratory

Sample Cohorts

Sample Type SM SMRNP SSA SSB JO-1 dsDNA

Positive 10 16 22 10 9 10

Negative 19 17 8 11 8 7

Y Y Y Y

Y

ANA 6-Plex Application Method► ANA specific microarrays – 16 pad, AVID slides with markers and controls

- Recombinant SM, SMRNP, SSA, SSB, JO-1 and Human dsDNA

- Positive controls – Anti-human IgG

- Calibration curves – Titrated Human IgG and IgM

- Intra-array normalizers – Recombinant Hep

Y

Y YY

Y Y Y Y

Y

YY Y

Y YY Y Y Y

Y

YYY

Incubate

Sample

Incubate

Primary AbWash

Wash

Y YY Y Y Y

Y

YYY Y YY

Y YY Y Y Y

Y

YYY Y YYY

Incubate

Detector/Probe

Wash

Image

AcquisitionData Analysis

45 Min. 45 Min. 45 Min. 1-2 Min.

Total Assay Time < 2 Hours

Analytica Approaches for Improved Assay Confidence

Analytical Methods Detection Controls/Calibrators

Level 1Standard Microarray /

Immunoassay MethodOne color - Emulates ELISA method

Level 2 Intra-Well Controls One color- Calibrators contained within sample well

- Enhanced confidence

Level 3 Intra-Spot Controls Multi-color

- Normalizer contained on sample binding

sites

- Highest confidence, most robust

► Level 1 Example - ANA 6-Plex Emulating ELISA Calibrators

Test Spots Sample

Control

Control Test Sample

1,000

RFU

5,000

RFU

Negative < RFU Cut-off < Positive

Anti-Human / Dye

Serum Sample IgG

Immobilized Ag

Anti-Human IgG

ControlTest Spots Sample

Control

► Level 1 - Analysis Results

+/- Determination cut-offs were derived from observed optimal

sensitivity/specificity results of sample testing

Level 1 – Analysis Results

Level 1 Analysis Results Based on Clinical Sample Characterizations Published

ELISA

ValuesSM SMRNP SSA SSB JO1 dsDNA

Sensitivity 63.6% 100% 100% 88.9% 100% 92.3% 8 – 70%

Specificity 66.7% 88.9% 100% 88.9% 100% 88.9% 87 – 100%

Level 2 - Intra-Well Calibrated 6-Plex ANA

Anti-Human / Dye

Serum Sample IgG

Immobilized Ag

Human IgG Control

(Titration Curve)

Anti-Human IgG Control

Test Spots Sample

ControlCalibration Curve

2,000

RFU5,000

RFU

Ratiometric Comparison

Negative < 0.8 – 1.2 < Positive

► Intra-well calibration provides improved control of assay

variability, improving sensitivity/specificity

Level 2 – Analytical Results

Level 2 Analysis Results Based on Clinical Sample Characterizations Published

ELISA

ValuesSM SMRNP SSA SSB JO1 dsDNA

Sensitivity 100% 100% 100% 100% 100% 100% 8 – 70%

Specificity 100% 92.9% 100% 100% 100% 100% 87 – 100%

Level 1 and Level 2 Comparison

SM SMRNP SSA SSB JO1 dsDNA

Level 1External Well

Calibration

Sensitivity 63.6% 100% 100% 88.9% 100% 92.3%

Specificity 66.7% 88.9% 100% 88.9% 100% 88.9%

Level 2Intra-Well

Calibration

Sensitivity 100% 100% 100% 100% 100% 100%

Specificity 100% 92.9% 100% 100% 100% 100%

Sensitivity Specificity

IF* 57 – 93% 41 – 63%

EIA/ELISA* 8 – 70% 87 – 100%

Multiplex Beads** 74 - 88% 78 - 97%

ArrayCAM Protein

Profiling System100% 93 – 100%

Grace Profiling System ANA 6-Plex Analysis Method Comparison

Comparison of Grace Profiling System ANA 6-Plex Compare to Other ENA Measurement Methods

* Source - Antinuclear antibodies and their detection methods in diagnosis of connective tissue diseases: a journey revisited, Diagnostic Pathology 2009,

** Source – Clin Vaccine Immunol. May 2007; 14(5): 505–509.

Absolute Sensitivity► Serially titrated serum sample was measured for four markers using RELISA® ENA testing kits

provided by Immuno Concepts, Inc.

► ELISA tests were read using a standard well plate reader provided by OSU/Cascades College

- LOD = Mean Blank + 2xSD

► Array shown to be more sensitive, primarily due to reduced noise at lower signal levels

Marker Array ELISA

SM <1:20,000 1:5,000

SNRNP <1:20,000 1:8,000

SSA <1:20,000 1:8,000

SSB <1:20,000 1:6,000

Approximate Sensitivity by Serum Titration

Layer 3 – Intra-Spot Multi-color Multiplex

Microarray Applications

Single Excitation wavelength and narrow emission

Wavelengths for Quantum Nanocrystals

Level 3 - Intra-Spot Multiplexing – Antibody Isotyping

► Further increased throughput

► Improved accuracy and consistency of two measurements for one sample

within one well

αHu IgM / Dye 1

αHu IgG / Dye 2

Human Sample IgM

Human Sample IgG

Immobilized Ag’s

Three Isotype ANA Screening Assay

IgG Detection

Q800IgM Detection

Q655

IgA Detection

Q585

Sample

B

Sample

A

Array Layout

Reactivity of Isotype controls

Hu IgG Controls

Hu IgM Controls

Hu IgA Controls

SSA

JO1

Anti-Hu IgG

Controls

JO1 JO1

JO1 JO1 JO1

Purified Rabbit IgG

Purified Mouse IgG

Purified Goat IgG

GAPDH

GSK

Akt

QDot 655

1. Goat anti-GAPDH +

anti-goat IgG QDot 585

2. Mouse anti-GSK +

anti-mouse IgG QDot 655

3. Rabbit anti-Akt +

anti-rabbit IgG QDot 800

3 Channel

Assay Detection

**

*

Level 3 – Intra-Spot Multiplexing for RPPA Normalization

Level 3 – Intra-Spot Multiplexing for RPPA Normalization

αMs IgG / Dye 1

αHu IgG / Dye 2

Mouse GAPDH IgG

Rabbit ERK1 IgG

Cell Lysate

► House keeping protein and marker of interest measured simultaneously within the same cell or tissue lysate spot for

optimal signal intensity normalization

► Data presented at RPPA Paris, October, 2014

Level 3 - Intra-Spot Normalization = Increased Assay Confidence

M H

αMs IgG / Dye 1

αHu IgG / Dye 2

Mouse IgG Cocktail

Human Sample IgG

Immobilized Ag’s

M

Control

Cocktail

(Sample

Diluent)

H

Human Test

Sample

► “Level Three Multplexing” provides assay controls

on the same binding site as the sample

M H

Marker 1

Calibrated Ratio = 2.0Marker 2

Calibrated Ratio = 1.0

Marker 3

Calibrated Ratio = 0.02

αMs IgG / Dye 1

αHu IgG / Dye 2

Mouse IgG Cocktail

Human Sample IgG

Immobilized Ag’s

Level 3 - Intra-Spot Normalization = Increased Assay Confidence

Level 1, Level 2 and Level 3 Reproducibility Comparison

► Reproducibility (%CV) for each assay analysis method

Assay MethodMean

Reproducibility

n = 8 n = 3

Inter-

array

Inter-

batch

Level 1 Net Signal 18.0% 18.3%

Level 2 Intra-Well Calibrated 14.0% 15.4%

Level 3 Two color – Intra-spot Calibrated 12.5% 12.7%

Conclusions

Platform Sens. Spec. Data OutputRepro-

ducibility

Through-

put

Controls and Assay Confidence

Absolute Sample

ControlsCalibrators

IF, REI 57-93% 37-63%Technician

SubjectiveNA Poor NA NA

ELISA 12-70% 87-99% Automated 10-20% CV Fair NA External, inaccurate

Bead Array 74-88% 78-97% Automated 10-20% CV Good NA External, inaccurate

ArrayCAM Proteome

Profiling System Standard63-100% 67-100% Automated 14.0% CV Excellent

-Sample control

- Assay controlsExternal, inaccurate

ArrayCAM Proteome

Profiling System with Intra-

Array Calibrators

93-100% 100% Automated 12.5% CV Excellent-Sample control

- Assay controls

- Intra-Well calibrated and

normalized– Robust

► Grace Bio-Labs Protein Profiling System

- Superior sensitivity and specificity

- Ease to use, non-subjective data outputs

- Excellent reproducibility

- High throughput

- Robust confidence of assay controls

- Overall low cost system

Questions?

► Please visit our booth during the Exhibition

► Or, to contact us:

www.gracebio.com