1. Development, Application and Validation of Q-Plex Technology

2. Presentation Outline Introduction Technology Case Studies
Case Study I. S. pneumoniae
Case Study II. Mycobacterium Bovis
Products and Services

3. 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 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

4. 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

5. 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 µm

6. What are Q-Plex Arrays?
PERFORMING THE ASSAY
Add 30 µl of sample
Wash
Add mix of detection antibodies
Add streptavidin bound to a IR-Dye or HRP

7. 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

8. 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

9. 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

10. 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

11. Plate Production Certified Clean Room (particle count <1000 ppm)
1-25 spots per well ranging from 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

12. 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)

13. 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

14. 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%
5.10%
4.70%
Assay #4
7.20%
7.30%
4.90%
4.50%
6.10%
7.10%
Assay #5
2.10%
3.60%
8.00%
2.30%
3.00%

15. Imaging - Chemi Chemiluminescent Detection via Strep-HRP
Quansys Q-View Imager
UVP EC BIOCHEMI
Kodak 4000MM
BioRad VersaDoc 4000 and XRS
Alpha Innotech HD2 or Fluorchem SP
Fuji LAS-3000

16. Imaging - IR IR- Fluorescent Detection via Strep-Dye LI-COR Aerius
LI-COR Odyssey

17. 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)

18. 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

19. 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

20. 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 :23-31

21. Case Study I: S. pneumoniae
Quansys & Luminex Comparison Data
R2 Values For Comparison to WHO ELISA
PnPs 4
PnPs 6B 9V 14
PnPs 18C
PnPs 23F
19F
Quansys to WHO
0.77
0.90
0.82
0.92
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 :23-31

22. Case Study I: S. pneumoniae
WHO Comparison Data 4 6B 9V 14
18C
19F
23F A 12 10 11
95% B 9
90% C 6
83% D 7 8
82% E
74%
ARUP-Luminex
73%
Quansys
88%
**American Journal of Clinical Pathology July :23-31

23. Case Study I: S. pneumoniae
Summary:
Average Quansys R2 values were 0.85, Average Luminex R2 values were 0.55.
The array correlated well with a standardized ELISA used for pneumococcal vaccine testing and with a calibration serum panel.
The array had good inter-assay and intra-assay reproducibility.
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 :23-31

24. 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

25. Case Study II: Mycobacterium Bovis
Test
TB (+)
Sensitivity (%)
TB(-)
Specificity (%)
ESAT-6
522
40.60
1489
86.60
CFP-1
82.60
69.70
MPB83
78.50
99.10
Anigen Lateral Flow
214
83.60 79
83.00
Enfer Multiplex
93.10
98.40
**Clinical and Vaccine Immunology Dec 2008

26. Case Study II: Mycobacterium Bovis
Summary:
Results allowed ENFER to find 13 markers of the original 20 with the highest diagnostic value for high throughput testing
Assay improved testing efficiency and costs
Assay allowed for rapid testing in centralized lab
**Clinical and Vaccine Immunology Dec 2008

27. Quansys Services
Sample Testing 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

28. Quansys Services

29. Q-Plex Array Selector

30. Quansys Products Human Mouse 16 Plex Cytokine Stripwell Kit
9 Plex Cytokine Inflammation Kit
16 Plex Cytokine Kit
9 Plex Angiogenesis Kit
9 Plex Cytokine IR Kit
16 Plex Cytokine IR Kit
Mouse
16 Plex Cytokine Kit
14 Plex Cytokine Inflammation Kit
9 Plex Cytokine IR Kit
16 Plex Cytokine IR Kt

31. Collaborative Products
MitoSciences MetaPath
Fatty Acid Array Plex
Mito Disease Array 4-Plex
LI-COR Infrarred Dye Technology
Mouse Cytokine - IR (9-plex)
Mouse Cytokine Screen - IR (16-plex)
Human Cytokine - IR (9-plex)
Human Cytokine Screen - IR (16-plex)

32. Publications **Many more on Website
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:
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.
“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

33. 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