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Development, Application and Validation of Q-Plex Technology

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.

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Development, Application and Validation of Q-Plex Technology

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  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 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

  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 350-500 µm

  6. 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

  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 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

  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

  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 2007 128:23-31

  21. Case Study I:S. pneumoniae Quansys & Luminex Comparison Data R2 Values For Comparison to WHO ELISA Quansys R2 average = 0.85 Luminex R2 average = 0.55 **American Journal of Clinical Pathology July 2007 128:23-31

  22. Case Study I:S. pneumoniae WHO Comparison Data **American Journal of Clinical Pathology July 2007 128:23-31

  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 2007 128:23-31

  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 **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.100+ markers to test 1 week turn aroundTested in triplicateData provided in report • Multiplex Array DevelopmentWide variety of technologies in houseProduce kits with lyophilized reagentsYears of experience in Array Development • Printing ServicesLiquid handling expertiseRobotics specialized in >10nl depositionPlates, slides, membrane, lateral flow, large volume dispense

  28. Quansys Services

  29. Q-Plex Array Selector

  30. Quansys Products • Human • 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 4-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 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 SerrorInfection 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

  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

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