development, application and validation of q-plex technology
TRANSCRIPT
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 pointsAcceptance 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
BioRad VersaDoc 4000 and XRS Fuji LAS-3000
Kodak 4000MMUVP EC BIOCHEMI
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
**American Journal of Clinical Pathology July 2007 128:23-31
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
Case Study I:S. pneumoniae
**American Journal of Clinical Pathology July 2007 128:23-31
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%
ARUP-Luminex 7 11 10 9 9 8 7 73%
Quansys 9 11 12 11 11 10 10 88%
WHO Comparison Data
Case Study I:S. pneumoniae
**American Journal of Clinical Pathology July 2007 128:23-31
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.
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
**Clinical and Vaccine Immunology Dec 2008
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
Case Study II:Mycobacterium Bovis
**Clinical and Vaccine Immunology Dec 2008
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
Summary:
Quansys Services
1. Sample Testing. 100+ markers to test 1 week turn aroundTested in triplicateData provided in report
2. Multiplex Array DevelopmentWide variety of technologies in houseProduce kits with lyophilized reagentsYears of experience in Array Development
3. Printing ServicesLiquid handling expertiseRobotics specialized in >10nl depositionPlates, slides, membrane, lateral flow, large volume dispense
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
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)
Collaborative Products
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. SidwellAntiviral 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