Dcninovatecpresentationfinal 101110124949-phpapp01

Complete, creative solutions for developers
and manufacturers of rapid assays
Complete, creative solutions for developers and manufacturers of rapid assays
Seminar on Business and Innovation in Biotechnology
INOVATEC
Belo Horizonte, Brazil. October 5th, 2010

• Brief introduction to DCN
• State of the art in applied rapid assay technology
– Standard formats for standard applications
– More advanced formats for quantification, high
sensitivity and digital interpretation
Agenda
• Market Opportunities and Market Demands
• Market Driven Innovation: Meeting market demand
– Materials and reagents
– Cassette and sample handler design
– Reader development
• System development case studies

Key Points
1. Rapid testing is a dynamically growing, readily accessible market
with opportunities available in numerous market segments
2. Assays can be developed to handle almost any reasonable market
condition, based on existing technology
– Simple, low cost lateral flow assays for simple, qualitative low
resolution applications
– More complex, more expensive lateral flow systems for high
– More complex, more expensive lateral flow systems for high
sensitivity, more quantitative, difficult applications
– These two types of assay are very different in how they are
developed and manufactured
– Other approaches to high performance rapid testing are
possible, but with a much longer outlook.
3. All of the elements needed to manufacture high performance tests
are available today, so while technological innovation is ongoing, we
can also choose to innovate in terms of where we apply
these technologies

Introduction to DCN
• Based in Carlsbad, California
• Founded in 2003 with the purpose of assisting companies in
developing, manufacturing and commercializing rapid tests for
any application.
• Our primary platform technologies are lateral flow, flow
through, microfluidic, dipstick (chemistry) and rapid ELISA,
using visual, fluorescent or paramagnetic detection systems
• We work in any market segment where these technologies can
be applied

Primary Services and Products
Rapid assay development and manufacturing
From Feasibility and Prototyping to full Development and
Manufacturing Transfer
Education and training courses in rapid assay technology
Learn how to develop and manufacture rapid tests
Custom conjugation services
protein – particle, protein-protein, small molecule etc
Consulting
on any aspect of rapid assay development
and commercialization
Products
Materials starter kits
Gold conjugation kits
Latex conjugation kits
Colloidal gold
All related lateral flow materials
Lateral flow reader systems: Fluorescent and visual

• Fully equipped for the manufacture of lateral flow, flow through,
and other rapid assay formats
• 8000 square feet, containing 3000 square feet of wet lab, housed in
a class 10,000 capable cleanroom, and an associated controlled
humidity room
• ISO 9001:2008 compliant, with EN 13485 by end 2010
Facility and Personnel
• ISO 9001:2008 compliant, with EN 13485 by end 2010
• Development staff of 14 industrially experienced assay development
scientists
Reagent Prep Area
Main Lab (Class 10,000 capable
cleanroom)
Component
Processing Area
(Temperature /
Humidity
Controlled)

Our Markets
Medical Diagnostics
Veterinary Diagnostics
Agricultural /
Environmental
Food Testing
OTC/Consumer
Our market is
international and
spread over multiple
application areas.
OTC/Consumer
Pharmaceutical
Biowarfare
Law enforcement /
Forensics
Microbiology
Nucleic Acid Testing
Industrial Health
and Safety
Approximately 40% of
our assay development
programs are US-
based, 40% are
European and 20% are
spread among Asia and
Latin America

Representative Clients
• Johnson and Johnson (UK)
• Novozymes (Denmark)
• Amic (Sweden)
• ICP Bio (Australia)
• Rockeby (Australia)
• Government of Thailand NIH (Thailand)
• Pacific Biotech (Thailand)
• BioRad (US/EU)
• Chiron Corporation (US)
• Amgen (US)
• Abbott (US)
• Genzyme Diagnostics (US)
• Becton Dickinson (US)
• Church and Dwight (US)
• Pacific Biotech (Thailand)
• Span Diagnostics (India)
• Daiichi Fine Chemical (Japan)
• Elixir Biosciences (China)
• MARS/Masterfoods (UK)
• Laboratorio Silanes (Mexico)
• Mintek (South Africa)
• Biosensia (Ireland)
• Church and Dwight (US)
• 3M (US)
• Whatman /GE (US / Europe)
• Aspen BioPharma (US)
• Genetic Testing Institute (GTI) (US)
• Cibavision (US)
• CDC (US)
• NIH (US)

Simple Devices for Rapid Testing
Standard Lateral Flow Strip
Sample addition
Conjugate
solubilization
Capillary flow;
antibody binding
Membrane clearing;
optical reading
Step 1
Step 2
Step 3
Step 4

Simple Devices for Rapid Testing
Standard Flow – through device
Sample addition
Optional wash
Conjugate addition; antibody binding
Final wash; membrane clearing;
optical reading

Evolution in Point of Care Assays
Drivers for Innovation and Market Growth
• Early POC devices were designed for qualitative applications
• Earliest applications in the 1980s were pregnancy tests,
growing in the 1990’s to include drugs of abuse and
infectious disease tests
• Market has grown steadily at a minimum CAGR of 10% since
the late 1990’s
• Market demand for tests in non-traditional areas has
expanded to include quantitative, high sensitivity
applications using difficult matrices, driving development
• Intellectual property issues have driven technological
innovation strongly in the past decade

Innovative Applications

Why lateral flow assays dominate the
(non glucose) POC market
• Ease of use: Minimal operator dependent steps and
interpretation
• Can handle small volumes of multiple sample types
• Can have high sensitivity, specificity, good stability
• Good Manufacturability and Scalability
• Good Manufacturability and Scalability
• Minimal market and user education required
• Market presence and acceptance
• Short development cycle – once reagents are developed, the
timeline to manufacturing transfer and approval is short
relative to other biotech products

• Sensitivity
• Control of user interpretation
• Reproducibility (CV)/ Quantification
• Multiplexing is difficult
Typical performance challenges in standard rapid assays

Application Requirements are Driving the
Technology Forward
•The requirements that the major rapid test markets are demanding
include:
• Speed to result: 2-10 minutes
• Sensitivity: high picogram/ml to low nanogram/ml
• Ability to handle difficult sample matrices
• Quantification
• Quantification
• Multiplexing
• Removal of interpretation issues
• Control of data

Evolution in Rapid Test Technology:
An Exercise in Market Driven Innovation
Clients provide use
inspired basic research
Pasteur's Quadrant describes scientific research that both seeks
fundamental understanding of scientific problems, and, at the same
time, seeks to be eventually useful.
Development in rapid diagnostic testing incorporates elements of use
inspired basic research (application development) and pure applied
research (technology development)
DCN and partners provide
the applied element

Bridging the Concept Gap
DCN
• Product Requirements Development
• Concept Refinement
• Feasibility and prototyping of all system elements
• Product development and system integration
• Transfer to manufacturing
• Commercialization

Veterinary
9
Food and Beverage
1
Pharmceuticals
0.6
Envionmental
0.2
Water
0.2
Medical Diagnostics
Veterinary
Food and Beverage
High Performance Allows for New Applications
Rapid Test Market Penetration and Opportunities
Medical Diagnostics
89
Percent of Market by Application
Total Market 2007: USD $2.3 billion
Total Market 2012: USD $$3.7 billion
Pharmceuticals
Envionmental
Water

Major Rapid Test Market Opportunities
1. Medical Diagnostics
35%
20%
21%
13%
7% 4%
% market 2007
Pregnancy Infectious diseases Cardiac markers
Cholesterol/lipds Drugs of abuse Other
•Growth opportunities exist in all
medical segments
•Growth will be driven by
reimbursement in US and EU markets
20%
24%
27%26%
15%
5% 3%
predicted market 2012
Pregnancy Infectious diseases Cardiac markers
Cholesterol/lipds Drugs of abuse Other
•Requires performance improvements
in quantification, sensitivity,
reproducibility and multiplexing

2. Veterinary Diagnostics
• Market was $205 million (USD) in 2007
• 9% CAGR to 2012 predicts a $310 million market by 2012
• Highly fragmented, price sensitive market, split among small
animals and livestock
• Major segments are infectious disease testing, metabolic
testing (cancer), diabetes and fertility/pregnancy
•Small animal tests tend to be paid for by owners and are non-
regulated for the most part. Large animal testing tends to be
government mandated and is regulated
•Growth driven by acceptance in large animal testing for
infectious disease and fertility, and control of antibiotic use and
in small animals by cancer and infectious disease testing
•Requires performance improvements in quantification,
sensitivity, reproducibility and multiplexing

3. Food and beverage testing
• Worldwide market of about $30 million in 2007
•Expected CAGR of 15% predicts $60 million by 2012
•Market is under-penetrated by rapid tests due to the
preponderance of microbiological testing (slow, traditional
technology largely used)
•Combinations of molecular and immunological testing may be
important to this market
•Growth will be driven by increased awareness of rapid
formats coupled with sampling and performance
improvements that can generate appropriate sensitivity

4. Water Testing and Environmental Remediation
• Worldwide market of about $5 million in 2007.
• Expected CAGR of 4% predicts $6 million by 2012.
• Highly regulated markets, with local or governmental agencies
setting requirements for testing, often in association with AOAC
(Association of Official Analytical Chemists International)
• Most testing is performed in certified laboratories by approved
methods
methods
• Rapid tests are typically performed as spot checks between
mandated test periods
• Also used for household and industrial testing of water quality in
certain markets
• Growth will be driven by increased awareness of rapid formats
coupled with performance improvements

• Option 1: Develop completely new platform technologies
based on microfluidics and alternative detection technologies
e.g. lab on a chip,
Market – Driven Innovation
Making test performance meet market demands
• Option 2: Improve the existing technology platform

A System Approach
• Rapid lateral flow and flow through assays can meet many
of the market’s requirements, but a different approach to
the assay is needed
• High performing rapid assays need to be approached in the
same way as high performance laboratory based systems
• Key system elements:
• Key system elements:
1) A properly developed assay made using
1) High quality materials
2) High quality reagents
3) Highly sensitive label for sensitive analyte detection
2) A well designed sample device and housing for the test
3) A controlled method for result interpretation
4) A well designed and controlled manufacturing process

1. Assay
Materials
• Analytical procedures usually require multiple steps
• Fluidics minimize number of steps in a test and increase ease of use
• In most POC devices these steps are performed using treated porous
matrices
• Fluid flow in POC devices usually relies on capillary flow
• Fluidic elements are usually designed to be disposable, and are
• Fluidic elements are usually designed to be disposable, and are
therefore made from low cost materials
• Suppliers must be of the highest quality. Lowest cost materials will not
support higher specified applications
Test Line
Control Line
Particle Conjugate
on conjugate pad
Wick
Sample Pad
Nitrocellulose MembraneBacking

1. Assay
Reagents
• Recognition is generally Ab-Ag or Ab-Ab based
• Different characteristics are required for high performance in a
lateral flow system than in an ELISA, for instance, due to the
mechanics of the system:
– The method chosen for screening is important
• Important Parameters:
– Specificity
– Dissociation Constant (Kd)
– High Affinity (High Binding Rate Constant (kon))
– Antibody Class (subtype: IgG1, IgG2A, IgG3, etc.)
– Monoclonals typically preferred, but high purity
polyclonals can also be used effectively
7777

1. Assay
Labels
• Colloidal gold
– Typically 20-40nm
– Low cost, difficult to control passive conjugations, one color,
relatively sensitive. Most common in qualitative applications
• Latex: Colored, Fluorescent, Paramagnetic
– Typically 100-300nm
– Low cost, covalent conjugations, variety of chemistries, well
– Low cost, covalent conjugations, variety of chemistries, well
controlled, stable, variety of particles and labels. Most common for
high sensitivity, quantitative applications (fluorescent,
paramagnetic)
• Others
– Upconverting phosphors
– Colloidal carbon
– Direct labeling using fluorophores and enhancers

Commonly used fluorescent labels in POC Applications
• We love fluorescence for high end applications!
• DCN’s Experience:
– Dark Red Labels (660-680nm) give good results due to lack of
background fluorescence in biological fluids in that spectral area
– Europium: Highly sensitive and commonly used due to broad
Stokes’ shift and stability (300 – 625nm)
Stokes’ shift and stability (300 – 625nm)
– Direct labeling of antibodies using fluorescent dyes is also an
option – sensitivity an issue – requires customization of system
components.

2. Readers
The use of readers in lateral flow assay systems
Detection of the signal generated
by the reagents, translation into a
numerical or alphanumerical
output, and delivery of that
output
Use of readers allows for:
Negative Positive
100% Visually
Negative
100% Visually
Positive
Operator Dependent Negative or Positive
Use of readers allows for:
• Improved sensitivity
• Reduction in user interpretation
issues
• Data capture, analysis and
storage
• Connectivity
• Quantification
• New applications
Operator Dependent Negative or Positive
Critical features:
• Hardware
• Algorithms for data processing,
• Signal to noise reduction,
• Measurement and data distribution
• Calibration methods

Reader Supply Partners
• Off-the-shelf readers are available that can be readily modified
for any development program
• DCN represents two suppliers: LRE and Qiagen Lake Constance
(formerly ESE)
– Provide a base platform from which to start
– Have a history of development of this type of product
– Have experience of regulatory and technical requirements
– Can advise on important technical design considerations
– May be faster to market
– Generally have dedicated manufacturing capability
• Readers can be custom designed by experienced designer such
as Source Scientific, Polygenesis, Omnica (all DCN partners)
– Slower and more expensive route in return for a
custom solution

• Sensitive
• Affordable
• Simple to operate
• Small, Mobile and Robust
• Documented Results
• Objective Results
• Quantitative Results
ESE Quant
DCN’s Reader Partners: Handheld and Benchtop Readers
• Quantitative Results
DCN distributes both ESE and LRE readers
DCN acts as the assay integration partner for both suppliers for
customers worldwide
LRE POC Reader
0
200
400
600
800
1000
1200
1400
0 200 400 600 800 1000 1200 1400
Data points
Intensity[mV]
Scan1
Scan2
Scan3
Scan4
Scan5
Scan6
Scan7
Scan8
Scan9
Scan10
Scan11
Scan12
Scan13
Scan14
Scan15
Scan16
Scan17
Scan18
Scan19
Scan20
Control
Test

Comparison of gold and fluorescent labels
LF Tuberculosis Fluorescent Assay
• Based on a dark red dye (latex particle based)
• Monoclonal sandwich assay
• Compared directly to a colloidal gold method
• Results read digitally using the ESE-Quant reader
Dark Red Dye (Latex)
ex 660 em 680
0
20
40
60
80
100
120
500 550 600 650 700 750
Wavelength (nm)
Rel.Intensity
Excitation
Emission

LF Tuberculosis Fluorescent Assay
Gold vs. Fluorescent Latex
6
8
10
12
300
400
500
600
700
PeakHeight(pixelunits)
PeakHeight(mV)
Sensitivity in TB Protein Assay:
Gold vs Dark Red FlourescentLatex
0
2
4
0
100
200
0 0.1 0.5 0.75 1 1.25 5
PeakHeight(pixelunits)
PeakHeight(mV)
Concentration (ng/ml) FlourescentLatex Gold
• Sensitivity greater than 0.2 ng/mL.
• Using same antibody / antigen combination, the assay
developed using a Dark Red fluorescent particle is >10 fold
more sensitive than the assay developed using gold.

Anthrax Protective Antigen Assay
• Assay for a recombinant
Anthrax protein
• Quantitative &
qualitative results

2
3
4
5
6
7
8
9
10
11
Signal/NoiseRatio
Gold vs. Latex Comparison - Lateral Flow
Anthrax Protective Antigen Assay
Gold
Fluorescent
Anthrax Protective Antigen Results
0
1
2
1 10 100 1000 10000
Ng/mL rPA
• Fluorescent assay sensitive to
2 ng/mL. Approximately 60-
fold better sensitivity than
comparable gold assay
• Visual or reader based
results

3. Device Design
The importance of device design in test performance
• Cassette design plays an integral role in the
performance of the assay
• Early integration of cassettes with sample delivery
elements and with strips is a critical element of
the design process
the design process
• If a reader is to be used, design device with this
in mind – off the shelf cassettes will rarely work
• Novelty in approach can lead to major benefits
e.g. separation of sample handling and conjugate mixing from the
remainder of the device can significantly improve CV’s

The SymbioSysTm Program
• Rapid diagnostics have historically had less emphasis on
engineering than other industries. (ie. Medical Device)
• Typical approach: Develop the strip, then find or try to design a
cassette that works with it
– highly inefficient and counter-productive.
• With current demands for ease of use, precision, quantification,
• With current demands for ease of use, precision, quantification,
low cost of goods and reduced time to market, this method is
no longer acceptable.
• Concurrent engineering, assay development and user needs
development is required to achieve this level of performance.
• DCN and Symbient developed the SymbioSys program to
address this demand.

DCN’s Design Partner: Symbient Product Development
• Established in 2004.
• Focus on development of single use diagnostic devices.
• Product Types:
– Lateral Flow Cartridges
– Fluidic Cartridges with Integrated Functions
– Sample Delivery Systems - pipettes, lancet devices,
urine cups.
urine cups.
– Instrument Driven Systems

Services Provided
• Mechanical Product Design
• Industrial Design
• 3D Solid Modeling (Solidworks)
• Program Management
• Precision Prototype Machining
• Rapid Prototype Tooling and Molding
• LIM Silicone Molding
• Product Testing and Test Method Development.
CONFIDENTIAL
• Product Testing and Test Method Development.
• Failure Modes and Effects Analysis (FMEA)
• Process Development
• Manufacturing Transition

Sample Handling Device and Cartridge Design
• DCN and Symbient have
developed various
diagnostic cartridges
including single test strip,
multiple test strip and
specialty housings.
designed and developed
several devices for collecting
and processing samples for
diagnostic testing.
• Sample types include saliva,
whole blood, fecal matter,
urine, mucous and surfaces.
• Methods include integrated
buffers, mixing, filtering and
metering.

Device
Optical Reader
Example: Sample handling in a quantitative
agricultural test
• Vertical flow assay
• Rapid and quantifiable
• Increased sensitivity with
integrated pre-incubation
• Particulate samples do not cause
• Particulate samples do not cause
flow problems
1. PRE-INCUBATION
2. FLOW THROUGH
3. RESULT

Alpha Amylase Detection in Wheat
Hagberg Falling Numbers are determined by dropping a ball through a tube of
ground wheat. The time it takes in seconds for it to reach the bottom correlates
inversely to the amount of alpha-amylase in the wheat. High falling numbers
indicate low concentrations of alpha-amylase, and low falling numbers indicate a
high concentration of alpha-amylase.
Falling Number
0
50
100
150
200
250
300
350
400
srw170 srw138 srw139 cbh2.7 cbh2.8
FallingNumber
Wheat Sample
Comparison of Values from Device and True Falling
Number Values
True FN
DiagnostIQ Avg FN
DiagnostIQ Avg
[Amylase]
Falling Number
True
FN
avg
amyl
conc.
St
Dev %CV
304 1.54 6.4 1.77%
244 21.82 3.56 1.27%
223 36.02
16.8
4 6.77%
142 144.05 6.57 4.77%

Intellectual Property – Driven Innovation
Rapiplex: A Novel Multiplex Rapid Assay Platform
• RapiPlex: An integrated Point-of-Care in vitro diagnostic platform;
• Unique intellectual property position
• Multiplex, Quantitative and/or Qualitative, Versatile, Portable
• Three integrating technologies to provide a robust platform
• Fluidics
• Fluidics
• Immunodiagnostic assay systems
• Integrated fluorescent reade

+
Fluidic Chip
• Proprietary fluidic strip & chip not constrained by lateral flow IP
• Disposable oriented in the vertical position when an assay is run
• No overlapping capillary materials – gaps between materials
• Enables simultaneous multiplex analysis from a single sample
• Defined individual channels in which sample flow can be controlled
• Individual channel entry ports for each channel
• Flow control zone ensures accurate and controlled flow in each
channel
• Injection molded – High Impact Polystyrene
• Suitable for all assay formats and multiple biological fluid sample
types – demonstrated on saliva, serum and whole blood to date

400,000
500,000
600,000
700,000
800,000
Avg.PeakHeight(RFU)
Biosensia Myoglobin
Myoglobin assay
Representative DCN Data
0
100,000
200,000
300,000
0 50 100 150 200 250 300 350 400
Avg.PeakHeight(RFU)
Myoglobin Conc. (ng/ml)
Error Bars – 95% Confidence

150,000
200,000
250,000
300,000
Avg.PeakHeight(RFU)
Biosensia Myoglobin – Low End
Myoglobin assay
0
50,000
100,000
0 20 40 60 80 100 120
Avg.PeakHeight(RFU)
Myoglobin Conc. (ng/ml)
Error Bars – 95% Confidence

Myoglobin Conc. (ng/ml) 95% Confidence Limits
5 16,661 10,504
10 37,698 22,495
25 82,432 41,144
Myoglobin Assay
Current DCN Sensitivity Data
25 82,432 41,144
50 145,149 106,220
100 295,043 183,477
350 764,314 483,319

Myoglobin Dilution Series (N=12)
R² = 0.9985R² = 1
500,000
600,000
700,000
800,000
900,000
Avg.PeakHeight(RFU)
Biosensia Myoglobin
Before
After
0
100,000
200,000
300,000
400,000
10 100 1000 10000
Avg.PeakHeight(RFU)
log Myoglobin Conc. (ng/ml)
After
Poly. (Before)
Poly. (After)

Summary
• Solved major mechanical design issues
• Assay CV’s controlled
• Good Slope of Dose Response
• Clinically relevant sensitivity (~5 ng/ml)

Manufacturing Process Design
Membrane Conjugate Pad
Dispense test and control lines
Block membrane if necessary
Pre-treat pad
Dispense / dip conjugate
Sample Pad
Pre-treat pad
Dry
Dry Dry
DryDry
Assemble membrane, conjugate pad, sample pad, wick and backing into cards
Cut cards into strips
Assemble strips into cassettes
Package cassettes

Process Control
Batch Vs Continuous Processes
XYZ 3050 dispenser with
BioJets and AirJets
Dispensing Dipping Drying
BioJets and AirJets
Clamshell benchtop laminator
Manual Dip Tray
CM 4000 Guillotine Cutter
Lamination Cutting
Forced Air Oven

Equipment and Processes
Batch Vs Continuous
Dispensing, Dipping,
Drying
Reel-to-reel web handling and
dispensing platform
Lamination, Cutting

In-line processing provides for greater process control and equivalent
processing
In line camera and bad part
marking system on a BioDot
RTR 4500 inline dispensing
system
Equipment and Processes
Batch Vs Continuous
10
15
20
25
1
64
127
190
253
316
379
442
505
568
631
694
757
820
883
946
1009
1072
1135
1198
1261
1324
Pixels
Mean Pixel Width Std Dev %CV
17.57 0.89 5.1
Output from a quality control camera
system on a RTR 4500. Dispensing run
over a 50m dispense length, 50mm/s,
human IgG at 1mg/ml, 0.8ul/cm on
nitrocellulose

All the elements of an integrated device
are available over the counter
• High quality test development
• Reader development
• Device development
• Manufacturing equipment
• A partnering approach is generally required to bring
the specialized expertise and components into the
assay

DCN’s Network Partners
DCN’s Network of Affiliates is a group of closely knit companies
that work together to provide all of the key elements necessary to
design, develop manufacture and commercialize point of care
diagnostic systems. Member companies include:
• Manufacturing equipment suppliers
• Cassette and device designers
• High volume molding companies
• Materials converters
• Reader developers and manufacturers
• Contract manufacturers
• Platform technology suppliers

A reader based semi quantitative serological assay:
Rapid Assay for anti-PF4:Heparin in human plasma (HIT)
• Fluorescent, reader based, integrated RFID tag and novel cassette
• ESEQuant fluorescent reader from Qiagen Lake Constance
• Threshold assay, with Positive/Negative reporting
• Predicate ELISA in market. Rapid assay must be as sensitive.
• No known standards
• Intended for the professional clinical laboratory market
• Will be labeled as an IVD :developed under full design controls
• Will be labeled as an IVD :developed under full design controls
• Assay and device developed for large scale manufacturing

Major Challenges
• Highly charged specific molecule
• Total immunoglobulin assay imparts formatting requirement
– sample and conjugate cannot mix prior to interaction with
test line
• High sensitivity needed
• Lack of standards, so establishment of cutoff requires
comparison to performance of predicate ELISA =
comparison to performance of predicate ELISA =
Requirement for high correlation to ELISA result.
• Requirement for tight CVs
• Integration of novel device design elements for IP generation
• Predicate rapid assays in the field require multiple
additions, timing steps and have poor user interface. The aim
of the program is to generate a device without these
attributes

Comparative results of lateral flow and ELISA
data on all clinical samples
1000
1500
2000
2500
TestLinePeakHeight(RFU)
OD vs Test Line Peak Height
(All Data)
OD vs TL
0
500
1000
0 0.5 1 1.5 2 2.5 3 3.5 4
TestLinePeakHeight(RFU)
ELISA OD
OD vs TL
• A panel of 83 samples, characterized by predicate ELISA
• All samples came from patients who had received heparin
• Testing in triplicate / quadruplicate if adequate volume
• Average values shown.

Cutoff: Comparative
0.400
0.500
0.600
0.700
TL/CL
OD vs TL/CL (OD <1.0)
OD vs TL/CL
0.000
0.100
0.200
0.300
0 0.2 0.4 0.6 0.8 1
TL/CL
ELISA OD
OD vs TL/CL
0.3 od Cutoff

OD = 0.3
ELISA TL/CL TL
True positives 41 39 37
False Negative 0 2 4
True Negatives 42 42 42
False Positive 0 0 0
n 83 83 83
Collated Testing Results:
ELISA vs LFIA using various cutoffs
True Positive = Samples with OD > cutoff and detected as positive
False negative= Samples with OD > cutoff and detected as negative
True Negative= Samples with OD < cutoff and detected as negative
False Positive= Samples with OD < cutoff and detected as positive
n 83 83 83
PPV 100.00 100.00 100.00
NPV
100.00 95.45 91.30
Sensitivity
100.00 95.12 90.24
Specificity
100.00 100.00 100.00

Dilution Series: High positive Diluted in Negative
R² = 0.9975
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
TL/CLRatio
TL/CL Ratio Relative to ELISA OD, n=5
TL/CL ratio
Poly. (TL/CL ratio)
R² = 0.9989
-500
0
500
1000
1500
2000
2500
0.00 0.50 1.00 1.50 2.00 2.50 3.00
PeakHeight(rfu)
ELISA OD
Peak Test Line Height Relative to ELISA OD, n=5
5-14 serum
TL Ht
Poly. (5-14
serum TL
Ht)
0.0
0.00 0.50 1.00 1.50 2.00 2.50 3.00
ELISA OD

Integrated System Development
Using the DCN Network to develop complete assay systems
• Project Management
• System requirement definition
• Product specification
• Cassette design
• Rapid prototyping
• Mold design and development
• Reader development
• GUI design
• Reagent Development
• Assay design and development
• Regulatory consulting
• Quality system management
Program Setup
and
Management
System
Development
The client provides the innovation in reagents and application area
DCN provides the rest
• Mold design and development
• Mechanical engineering design
• GUI design • Assay design and development
• Calibration system development
• Documentation
• Manufacturing process design
• Quality system management
• FMEA / Risk analysis
• High volume plastic molding • Manufacturing equipment
supply
• Manufacturing facility setup
• Scale-up and manufacturing transfer
• Reagent manufacture
• Gap manufacturing
Commercialization
• Market research
• Board level advice
• Networking to finance
• Branding
• Troubleshooting
• Patent Landscaping
Manufacturing
Transfer

Key Points
1. Opportunities are available in numerous market segments for
rapid assays with the right level of performance
2. Assays can be developed to handle almost any reasonable
market condition, based on existing technology
– Simple, low cost lateral flow assays for simple, qualitative
low resolution applications
– More complex, more expensive lateral flow systems for
– More complex, more expensive lateral flow systems for
high sensitivity, more quantitative, difficult applications
– These two types of assay are very different in how they
are developed and manufactured
– Other approaches to high performance rapid testing are
possible, but with a much longer outlook.
3. All of the elements needed to develop and manufacture
high performance tests are available today

Acknowledgements
• DCN
– Roy Chung
– Jerrold Loo
– Wendy Mendiola
– Michael Fiechtner
• Symbient Product Development
– John Zeis
– John Zeis
– Scott Castenon
• Shara Rosen for marketing information
• Thanks to GTI and Biosensia for use of data
• Thanks to Cynthia Rocha, Camila Resende Duarte,
Gislaine Silva, Regina Fernandes from SECTES and
Christiane Contigli from CETEC and all who
helped to coordinate my attendance at
INOVATEC

www.dcndx.com
Contact :
Brendan O’Farrell at
bofarrell@dcndx.com
Diagnostic Consulting Network Inc
6354 Corte del Abeto, Suite B-C
Contact Information
Please email to receive copies of the slides
6354 Corte del Abeto, Suite B-C
Carlsbad, CA 92011
Tel: 760-804-3886
Fax: 760-804-3866

• Quickly develop and commercialize rapid assays for any application
• Experienced with fluorescent, visual (colloidal gold / latex) and
paramagnetic labels
– From concept to commercialized assay in under 12 months
We can develop assays for you:
Assay Development Services
– From concept to commercialized assay in under 12 months
– ELISA conversion to rapid assay formats in short timeframes
– Reagent sourcing and development
– Rapid prototyping
– Feasibility studies
– Complete development
– Manufacturing transfer

• The plastic devices used in these assays can determine their ability to
perform in demanding applications and need to be custom developed
in virtually any high performance application
• DCN partners with product designers, such as Symbient Product
Development and molding companies such as HK Plastics to integrate
We can design and develop your plastic devices:
Cartridge and Device Design Service
new cassettes and novel device designs into programs for clients
• Partners can be contracted through DCN, and DCN will manage the
entire development program on behalf of the client

• DCN partners with the major suppliers of readers to the point of care
marketplace to provide reader solutions for our customers.
• We can provide readers for:
– Visual labels
– Fluorescent labels
– Paramagnetic labels
We can help you develop reader systems for your assays
Reader Development and Integration
– Paramagnetic labels
• Our primary partners for off the shelf and custom reader supply are QLC
(formerly ESE) and LRE (Esterline), and we distribute for both companies in the
US.
• We also work with custom reader design and development houses including
Source Scientific and Polygenesis.

• DCN can develop and
commercialize all aspects of
a point of care diagnostics
platform, through our in-
house capabilities and our
Network of Affiliates
We can design and develop your complete assay system
Integrated System Design and Development
Cartridge top housing
RF id tag
Cartridge bottom housingStrip and Assay
• Custom design and
integration for high
performance assays our
specialty
Desiccant

We can connect you to all of the best suppliers:
The Network of Affiliates
DCN’s Network of Affiliates is a group of closely knit companies
that work together to provide all of the key elements necessary to
design, develop manufacture and commercialize point of care
diagnostic systems. Member companies include:
• Manufacturing equipment suppliers
• Cassette and device designers
• High volume molding companies
• Materials converters
• Reader developers and manufacturers
• Contract manufacturers
• Platform technology suppliers

• Learn how to develop and manufacture lateral flow assays
• Customized one-on-one courses held at DCN’s core facility in
Carlsbad, California
• Scheduled on demand
• Duration 3 – 5 days
We can teach you to develop and make your own assays
Education and Training Services
• Theoretical and practical elements dealt with
• Bring your reagents and optimize an assay during the course

We can help with any aspect of commercialization
Consulting Services
DCN has over 20 consultants on staff that can provide a wide range of
services related to the point of need diagnostics or detection business:
• Regulatory Affairs
• Project management
• Risk assessment
• Intellectual property landscaping
• Intellectual property landscaping
• Manufacturing process development
• Assay troubleshooting
• Quality system establishment for diagnostic companies
• Device design
• Facility setup
• Equipment specification and procurement
• Market evaluation
• Technology evaluation

• DCN’s facility is completely equipped for manufacture of lateral flow, flow through, and
other format point of need assays
• The facility:
– 8000 square feet, containing 3000 square feet of wet lab, housed in a class 10,000
capable cleanroom, and an associated controlled humidity room
– Currently ISO 9001:2008 compliant and will complete EN 13485 by end Q4 2010
• Manufacture non-FDA regulated product for several clients in the US and Europe, and
We can manufacture your tests for you
Contract Manufacturing
components and reagents for clients in the US and outside
• DCN partners with several dedicated contract manufacturers to whom
it can transfer product for manufacturing.
Reagent Prep Area
Main Lab (Class 10,000 capable
cleanroom)
Component
Processing Area
(Temperature /
Humidity
Controlled)

• DCN provides everything needed to develop or manufacture lateral flow or
flow through assays
– Colloidal gold, colored and fluorescent latex beads and conjugates
– Assay materials: membranes, pads, backing, cassettes wicks, etc
– Antibodies and antigens
– Small molecule conjugates
We can supply you with key components
Materials, Reagents and Kits
– Small molecule conjugates
– Test strip readers: Handheld and benchtop
• Kits of materials and reagents
– Colloidal gold
– Material Starter Kit
– Colloidal gold conjugation kit
– Latex conjugation kit
– Gold conjugation kit

We can make the critical reagents for you
Custom Conjugate Development and Manufacture
• Expertise in covalent conjugations
– Protein / peptide /small molecule –Particle
• Colored, fluorescent or paramagnetic latex, gold, quantum dots,
etc.
– Protein – Protein
Streptavidin Bead #2
2.5
Strep #2 (1X)
• Full optimization process
• Full QC including binding
assays where reagents are
available
• For diagnostics or separations (magnetic latex)
• Small to full prep scale
-0.5
0
0.5
1
1.5
2
0 5 10 15 20 25
ng Biotin
OD
Strep #2 (1X)
Strep #2 (10X)
Strep #2 (20X)
Merck Strep REC/1.0um
Candidat 2
Merck Strep C-REC/ 1.0um
Candidat 4
Merck Strep TREC/1.0um
Candidat 4
Merck Strep 0.2umCandidat 1

• Dr Roy Chung: Over 25 years of assay development and manufacturing
experience. Previously Lead Scientist at Nanogen
• Dr Hans Boehringer: Over 20 years of assay development and manufacturing
experience. Most recently Scientific Director at Genzyme Diagnostics
• Dr Brendan O’Farrell: 20 years of assay development, manufacturing,
equipment and manufacturing process development. Previously VP of
Technology Development at BioDot Inc and Director of R&D and Manufacturing
at SDS Inc.
Our People
at SDS Inc.
• Michael Surmanian: 20 years of biotech technology development experience.
Formerly President of BioDot Inc.
• Assay Development Team of 10 scientists, all of whom worked for major
human and veterinary diagnostic companies. Minimum of 5 years of experience
Consulting Group.: Over 25 industry experts available. Examples:
• Dr Judith Howard: Ex VP of Quality, Regulatory at Inverness Medical
Innovations
• Dr David Boudreau: Ex Director of Operations, Nanogen Inc
• Michael Weinholt: President of Regulatory Consulting Inc

Our Strengths
• Expertise in reagent development, assay development, device design
and reader integration
• Experience in all aspects of system development, regulation,
commercialization and manufacturing
• Modular menu of services, allowing for flexible project execution –
• Modular menu of services, allowing for flexible project execution –
select only the services you need
• Parallel development strategies for device and assay development –
science and engineering combined
• Networked to all of the partners you will need to get to market
• Full design controls and documentation.
• Full support for registration activities
• Speed to commercialization

1) Assay
• Materials for fluid movement and reaction matrices
• Biological recognition reagents (antibodies/antigens)
• Signal generation reagents (labels)
2) Reader system
Challenges in Designing and Manufacturing
High Performance POC Assays
• Technology
• Supplier
3) Device
• Sample collection and processing
• Conjugate handling and mixing
• Cassettes
4) Manufacturing process design

Market Penetration by Application
25
30
35
40
45
% of Available Market, 2007
(compared to lab based immunoassays that have a rapid segment)
0
5
10
15
20
Medical
Diagnostics
Veterinary Food and
Beverage
Pharmceuticals Envionmental Water

60
70
80
90
Percentage of Available Market by
Geographical Region
Market Penetration by Geographical Region
0
10
20
30
40
50
US
EU
ROW

• Sensitivity:
• Fluorescence based tests can be more sensitive than colorimetric
• Higher sensitivity allows new applications and markets
• Control
• Fluorescent assays are based on latex particles which can be
Why we like fluorescent labels
for reader based applications
coupled covalently. Colloidal gold conjugation is passive.
• This allows for better control of the conjugates and improved CV‘s
• Availability
• High quality, high performance beads available
• Multiple sizes, chemistries
• Availability of off-the shelf readers
• Makes development easy and low cost
• Makes fluorescence a comparatively open system

Device Design Trends and Implications
• IP considerations have driven a trend towards novel device design:
– clear pathways to designing non-infringing devices exist
• For many applications, sample handling, sample preparation,
extraction and delivery to devices need to be performed
– E.g. Tests for agriculture, biowarfare, food pathogen testing,
environmental testing involve handling difficult samples, treating
them and delivering them to devices in difficult field conditions.
– Devices can be designed and integrated to cassettes to perform
these functions

Myoglobin Conc.
(ng/ml)
n
Avg Peak
Height
CV
0 15 0 na
5 15 13,583 11.3%
10 15 30,097 12.6%
Myoglobin assay data
10 15 30,097 12.6%
25 15 61,788 16.7%
50 15 125,685 7.7%
100 15 239,260 11.7%
350 15 623,817 11.3%

Prototyping Capabilities
CNC Machining Steriolithography (SLA)
CNC Machining
Prototype Mold Fabrication Injection Molding
Steriolithography (SLA)

Assembly and Testing

Myo Conc. (ng/ml) N Avg. TL pk ht TL %CV
25 12 64,948 11.0%
50 12 118,659 5.9%
Myoglobin Dilution Series (N=12)
100 12 229,002 10.4%
250 12 457,563 4.1%
500 12 749,168 6.9%

Overall Conclusion
• Doing so requires careful design of all aspects of the
product and the integration of high quality fluorescent
label, cassette and reader technologies
• The design and development of products of this nature is a
highly specialized task
• That is where a company like DCN, with specialist
knowledge and experience, comes into the picture
• How can we help you?........
• How can we help you?........

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