Development, Application and Validation of
Q-Plex Technology
Presentation Outline
Introduction
Technology
Case Studies
•Case Study I. S. pneumoniae
•Case Study II. Mycobacterium Bovis
Products and Services
Quansys Background
Quansys Biosciences develops cutting edge multiplexing technologies and
processes to improve the accuracy, simplify the process and reduce the time and
expense of ELISA testing.
•June
2005 - Quansys Biosciences formed to commercialize the multiplex
ELISA (Q-Plex™) technology
•Technology used in many markets – agriculture, diagnostics, research and
development
•Wholly owned subsidiary of the non-profit Spendlove Medical Research
Institute which was founded by Dr. Rex Spendlove (founder of Hyclone Lab.)
•Royalty payments back to the Research Institute to fund further research
Q-Plex™ Array
•Simultaneously measure
multiple (up to 25) proteins
•Saves sample, time and
money
•“Bridging Technology”
•High Specificity
•Low LLDs
•Quantitative
•Low Sample Volume
•Up to 25 assays/well
•One Spot = One Assay
•Customizable
•Automated Analysis
Software
What are Q-Plex Arrays?
ANTIBODY DEPOSITION IN 96
WELL PLATE
•Robotic liquid handlers print
20-50nl spots of capture
antibody
•Each spot is a unique assay
within the well
•Spot to spot CV <4%
•Spot size 350-500 µm
What are Q-Plex Arrays?
PERFORMING THE ASSAY
•Add 30 µl of sample
•Wash
•Add mix of detection
antibodies
•Wash
•Add streptavidin bound to a
IR-Dye or HRP
What are Q-Plex Arrays?
DETECTION OF ANTIGEN IN
SAMPLE
•Each spot is a different
assay (IL-1, IL-2, etc.)
•With the addition of
substrate, a response is
produced
•If antigen is present the spot
emits light
•If no antigen is present the
spot is not visible
What are Q-Plex Arrays?
IMAGE CAPTURE
•An image is taken of the
plate via high resolution
camera or fluorescent
scanner
•The image file (TIFF) is
imported into Q-View
Software
What are Q-Plex Arrays?
IMAGE ANALYSIS
•Image is opened in Q-View
Software
•User selects product
templates and specifications
•Spots are automatically
found on plate image
•Intensity of spot response is
measured and raw data is
generated
What are Q-Plex Arrays?
DATA ANALYSIS
•Raw data is analyzed and
compared to in-plate
standard
•5PL Regression models used
to calculate unknowns
•Standard curves are
calculated and sample and
statistical data is exported
Plate Production
•Certified Clean Room
(particle count <1000 ppm)
•1-25 spots per well ranging
from 50-10 nano-liters
•High Throughput
Production Capabilities
•Quality Assurance of
Printing
•Capture agent spotted
with dye
•Plates imaged with
CCD imager
•Spots characterized to
ensure proper spotting
Kit Production
•Lyophilized temperature
sensitive materials
•Long term stability
•1 year
•Bulk packaging available
•Large batch productions
•Thorough instruction
manual
•Video tutorials available
(You Tube and on website)
Quality Control
ISO 9001:2000 Registered
Quality Policy:
Quansys Biosciences is committed to exceptional customer
service, high quality products and developing employees while
complying with and continually improving the quality management
system.
Each kit is QC at 192 different points
Acceptance criteria for LLD, Standard curves, background, stability
and many other factors
Quality Control Variation
Intra and Inter Plate %CV
Plate #1
Plate #2
Plate #3
Plate #4
Plate #5
Plate #6
Plate #7
Plate #8
Assay #1
3.20%
4.40%
1.90%
2.90%
3.80%
3.70%
3.10%
4.00%
Assay #2
3.90%
5.70%
4.60%
5.40%
3.50%
3.40%
2.20%
4.80%
Assay #3
3.30%
2.50%
4.80%
5.10%
3.80%
3.40%
3.70%
4.70%
Assay #4
7.20%
7.30%
3.50%
4.90%
4.50%
6.10%
4.60%
7.10%
Assay #5
2.10%
3.60%
8.00%
2.30%
3.00%
2.50%
4.00%
4.80%
Imaging - Chemi
Chemiluminescent Detection via Strep-HRP
Quansys Q-View Imager
Alpha Innotech HD2 or
Fluorchem SP
Kodak 4000MM
BioRad VersaDoc 4000 and
XRS
UVP EC BIOCHEMI
Fuji LAS-3000
Imaging - IR
IR- Fluorescent Detection via Strep-Dye
LI-COR Odyssey
LI-COR Aerius
Imaging Support
Imager Tutorials and Manuals
•
Online Imager Webpages
• (Quansys Website)
•
Tech Support for configuring customer imagers
• (Quansys Tech Support)
•
Videos on Imagers
• (visit Quansys Website or search Quansys on YouTube)
Imaging – Q-View™
•
High Resolution Digital Imaging
System (15MP)
•
Optimized for imaging arrays
using Q-Plex™ technology
•Range Enhancement (4-5 log)
•Vignetting Correction
• 12 to 2% CV
•
Chemiluminescence Western
blot imaging
•
Automated image acquisition
and analysis software with PC
•
Low Cost
Software
Q-ViewTM Software
•Automated Image Capture
from Quansys Imager
•Image Processing
•Auto Spot finding
•Well and Sample Assignment
•Data Analysis (5PL)
•Comprehensive Reports
•Free version auto spot finds
and outputs raw data. 20 day
evaluation of full software
Case Study I:
S. pneumoniae
Collaboration with ARUP Laboratories
Inc. Salt Lake City, Utah
Testing for antibodies to each of the
different serotypes of Pneumococcus
Tested standardized Goldblatt samples
in comparison to Luminex and WHO
standardized ELISA
Validation performed at Quansys and
ARUP with different technicians
Specs: Custom Software and Imager
built
Rapid Assay Time: 15 minute array
**American Journal of Clinical Pathology July 2007 128:23-31
Case Study I:
S. pneumoniae
Quansys & Luminex Comparison Data
R2 Values For Comparison to WHO ELISA
PnPs
4
PnPs
6B
PnPs
9V
PnPs
14
PnPs
18C
PnPs
23F
PnPs
19F
Quansys to
WHO
0.77
0.90
0.82
0.92
0.90
0.69
0.97
Luminex to
WHO
0.71
0.44
0.60
0.89
0.09
0.20
0.95
Quansys R2 average = 0.85
Luminex R2 average = 0.55
**American Journal of Clinical Pathology July 2007 128:23-31
Case Study I:
S. pneumoniae
WHO Comparison Data
4
6B
9V
14
18C
19F
23F
A
12
12
10
12
11
12
11
95%
B
11
12
12
10
11
9
11
90%
C
9
6
11
12
11
9
12
83%
D
7
11
12
9
8
12
10
82%
E
11
7
9
8
11
7
9
74%
ARUPLuminex
7
11
10
9
9
8
7
73%
Quansys
9
11
12
11
11
10
10
88%
**American Journal of Clinical Pathology July 2007 128:23-31
Case Study I:
S. pneumoniae
Summary:
I.
Average Quansys R2 values were 0.85, Average Luminex R2 values
were 0.55.
II.
The array correlated well with a standardized ELISA used for
pneumococcal vaccine testing and with a calibration serum panel.
III.
The array had good inter-assay and intra-assay reproducibility.
IV.
The array would reduce the time, resources and cost over the current
method of testing each serotype individually by ELISA.
**American Journal of Clinical Pathology July 2007 128:23-31
Case Study II:
Mycobacterium Bovis
Performed at Enfer Group Ltd., Dublin, Ireland
Tested 20 different proteins and peptides associated with Bovine TB
Tested 1489 negative samples and 522 positive samples
Tested against single ELISAs assays (ESAT-6, CFP-20 and MPB83) and
Tested against single lateral flow assay (MPB83)
Custom development and printing from Quansys
**Clinical and Vaccine Immunology Dec 2008
Case Study II:
Mycobacterium Bovis
Test
TB (+)
Sensitivity (%)
TB(-)
Specificity (%)
ESAT-6
522
40.60
1489
86.60
CFP-1
522
82.60
1489
69.70
MPB83
522
78.50
1489
99.10
Anigen Lateral Flow
214
83.60
79
83.00
Enfer Multiplex
522
93.10
1489
98.40
**Clinical and Vaccine Immunology Dec 2008
Case Study II:
Mycobacterium Bovis
Summary:
I.
Results allowed ENFER to find 13 markers of the original
20 with the highest diagnostic value for high throughput
testing
II.
Assay improved testing efficiency and costs
III.
Assay allowed for rapid testing in centralized lab
**Clinical and Vaccine Immunology Dec 2008
Quansys Services
1.
2.
3.
Sample Testing.
100+ markers to test
1 week turn around
Tested in triplicate
Data provided in report
Multiplex Array Development
Wide variety of technologies in house
Produce kits with lyophilized reagents
Years of experience in Array Development
Printing Services
Liquid handling expertise
Robotics specialized in >10nl deposition
Plates, slides, membrane, lateral flow, large volume dispense
Quansys Services
Q-Plex Array Selector
Quansys Products
Human
Mouse
•16 Plex Cytokine Stripwell Kit
•16 Plex Cytokine Kit
•9 Plex Cytokine Inflammation Kit •14 Plex Cytokine Inflammation Kit
•16 Plex Cytokine Kit
•9 Plex Cytokine IR Kit
•9 Plex Angiogenesis Kit
•16 Plex Cytokine IR Kt
•9 Plex Cytokine IR Kit
•16 Plex Cytokine IR Kit
Collaborative Products
MitoSciences MetaPath
•Fatty Acid Array
•Mito Disease Array
4-Plex
4-Plex
LI-COR Infrarred Dye Technology
•Mouse Cytokine - IR (9-plex)
•Mouse Cytokine Screen - IR (16plex)
•Human Cytokine - IR (9-plex)
•Human Cytokine Screen - IR (16plex)
Publications
1-
“Validating a custom multiplex ELISA against individual commercial immunoassays using clinical samples."
Michael Liew, Matthew C. Groll, James E. Thompson, Sara L. Call, Joann E. Moser, Justin D. Hoopes, Karl Voelkerding, Carl Wittwer, Rex S. Spendlove Biotechniques, February 2007
2-
TLR3 Deletion Limits Mortality and Disease Severity due to Phlebovirus Infection.
Brian B. Gowen, Justin D. Hoopes, Min-Hui Wong, Kie-Hoon Jung, Kevin C. Isakson, Lena Alexopoulou, Richard A. Flavell, and Robert W. Sidwell - The
Journal of Immunology, Nov 2006, 177: 6301-6307.
3-
Enhancement of the infectivity of SARS-CoV in BALB/c mice by IMP dehydrogenase inhibitors, including ribavirin.
Dale L. Barnard, Craig W. Day, Kevin Bailey, Matthew Heiner, Robert Montogomer, Larry Lauridsen, Scott Winslow, Justin Hoopes, Joseph K.-K. Li,
Jongdae Lee, Dennis A. Carson, Howard B. Cottam, Robert W. Sidwell
Antiviral Research Volume 71, Issue 1 August 2006, pages 53-63
4-
"A 22-plex Chemiluminescent Microarray for Pneumococcal Antibodies"
Authors: Jerry W. Pickering, Justin D. Hoopes, Matthew C. Groll, Heidi K. Romerol, Dave Wall, Howard Sant, Mark E. Astill and Harry R. Hill - American
Journal of Clinical Pathology
5-
“The effects of second-hand smoke on biological processes important in atherogenesis”
Hongwei Yuan, Lina S Wong, Monideepa Bhattacharya, Chongze Ma, Mohammed Zafarani, Min Yao, Matthias Schneider, Robert E Pitas, Manuela
Martins-Green - BMC Cardiovascular Disorders 2007, 7:1 (8 January 2007)
6-
“Exploring the Potential of Cytokine Arrays for Human and Mouse Cytokine Research” - Matt Groll
American Biotechnology Laboratory. July 2005, pg 25-26
7-
“Enterococcal Leucine-Rich Repeat-Containing Protein Involved in Virulence and Host Inflammatory Response”
Sophie Brinster, Brunella Posteraro, Hélène Bierne, Adriana Alberti, Samira Makhzami, Maurizio Sanguinetti, and Pascale Serror
Infection and Immunity 2007 September; 75(9): 4463–4471.
8-
“Multiplex Immunoassay for the Serological Diagnosis of Mycobacterium bovis Infected Cattle”
Clare Whelan, Eduard Shuralev, Grainne O’Keeffe, Paula Hyland, Henry Kwok, Shane Olwill, Matt Groll, Sara Call,
Jim Johnston, Mary Jo Hamilton4, William C. Davis4 and John Clarke1* (In Press in Clinical and Vaccine
Immunology Dec 2008)
**Many more on Website
Quansys Biosciences
•“Bridging Technology”
•One Spot = One Assay
•Up to 25 Assays/well
•Quantitative
•High Sensitivity
•Low Cost
•Low Sample Volume Required
•ISO 9000/2001 Registered Company
•Quansys Biosciences: The high value leader