Serum Biomarker Chip. High Throughput Comparative Analysis of Known Serum Biomarkers

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1 Serum Biomarker Chip High Throughput Comparative Analysis of Known Serum Biomarkers Reproducibly pattern the relative abundance of 120 human serum biomarkers Panels of biomarkers related to early detection, benefits of therapy and likelihood of recurrence Sensitive two-color labeling and fluorescent detection Familiar genomic microarray chip format compatible with fluorescent scanners The Serum Biomarker Chip enables proteomics researchers to pattern the molecular signature of human serum. The chip addresses the need for high throughput technologies used to study and develop a range of research applications from risk stratification, to include disease prognosis, drug eligibility, prediction of safety and efficacy, and therapeutic monitoring. The Serum Biomarker Chip is a single antibody capture array built on the FAST Slide dual pad platform. Each slide has two identical arrays of antibodies printed in triplicate. Two-color fluorescent detection permits the researcher to reproducibly pattern the relative abundance of 120 human serum proteins between two samples, such as serum samples from diseased and healthy individuals. The Serum Biomarker Chip kit includes two arrayed dual pad slides, two Incubation Chambers, Protein Array Wash and Blocking Buffers. The slide holder and labeling/detection reagents are available separately. W H A T M A N S E R U M B I O M A R K E R C H I P D A T A S H E E T

2 Two-Color Labeling & Detection Kit The Two-Color Labeling and Fluorescent Detection Kit is designed to label two protein samples. The labeled proteins are pooled and probed against arrayed antibodies in a competitive binding assay, and detected using indirect fluorescence. Highly efficient and uniform labeling of complex serum samples Reproducible labeling and signal detection Stable, robust and fast non-enzymatic procedure Reduces ph dependency of labeling efficiency System solution includes labeling reagents, fluorescent conjugate and bench-friendly protocol The kit is intended for use with two-pad FAST Slides, including the Serum Biomarker Chip. The kit contains the Universal Linkage System (ULS ) chemistry to label samples containing approximately 250 µg of protein in serum, plasma, or a whole cell lysate. The kit is designed to label two different protein samples, each with a different hapten. Sufficient labeling reagent is provided to perform a hapten swapping experiment. Benefits of the two-color dye-swap assay Accounts for hapten-specific differences in either Biotin-ULS or Fluorescein- ULS labeling efficiencies Averages differences in antibodyantigen binding interactions caused by steric hindrance Minimizes chip-to-chip variability includes an internal control within the assay The first pad on the slide is probed with a mixture of two different protein samples, each labeled with a different hapten; the second pad is probed with the same two protein samples but with the haptens reversed. The normalized intensity for each element of each pad is calculated as the average of the biotin- and fluoresceinlabeled derived intensities from a two-pad experiment. The ratio between the signal intensity at each spot corresponds to the concentration ratio of the proteins found in the two samples. This method is attractive for antibody chips as it takes into account any hapten-specific differences in antigenantibody interactions. The use of the ULS labeling system minimizes background by using indirect fluorescence detection, labels multiple amino acids, and requires no additional materials or reagents. Labeling and Detection Procedure Overview Label serum proteins with Biotin-ULS ( ) and Fluorescein-ULS ( ) for 2-6 hours at 42 C Sample A Sample B Sample A Sample B Terminate reaction and remove unbound ULS reagent with spin columns Pool Samples Mix 1 Mix 2 Block slide with S&S Blocking Buffer for 30 min at room temperature Incubate labeled proteins with Serum Biomarker Chip for 4 hours to overnight at room temperature Wash 6X to remove unbound proteins W H A T M A N S E R U M B I O M A R K E R C H I P D A T A S H E E T

3 APPLICATION DATA Serum protein profiles of cancer patients. Serum proteins from cancer patients and corresponding age- and gender-matched controls were labeled with Biotin-ULS and Fluorescein- ULS, respectively, then pooled and probed against the Serum Biomarker Chip. The competitive binding was detected using both streptavidin-dy 647 and anti-fluorescein antibody-dy 547 conjugates. Spot-finding, background corrections, and two-channel data sampling was done using ArrayVision FAST software. The SBC Analysis Workbook is a Microsoft Excel file that converts fluorescent data into numerical values that represent the abundance of antigen in Sample A relative to Sample B. The average spot intensities for each analyte were plotted to reveal the over- and under-abundant serum biomarkers in the cancer patient (y-axis) compared to serum of a healthy individual (x-axis) for bladder, breast, colon, and prostate cancer. Upper and lower purple control lines represent 1.25 standard deviations above and below a ratio of 1 (no change in protein expression). Based on previous experimental results with the same serum labeled with both haptens and competed against itself, we recommend that only ratios greater than 1.25 std devs on either side of ratio=1 be considered significant expression level differences (these values lie outside the normal noise level of the assay). Ratios are plotted on a log 2 scale so that a value of 0 represents no change in expression between the 2 samples, with ratios representing up and down regulation being distributed equally around Bladder Cancer Breast Cancer Colon Cancer Prostate Cancer Excite 547 nm Excite 647 nm Prostate Cancer Incubate Serum Biomarker Chip with two-color fluorescent detection solution for 1 hour at room temperature Wash 6X to remove unbound proteins Scan at two wavelengths, spot-find, and sample data Bioinformatic analysis W H A T M A N S E R U M B I O M A R K E R C H I P D A T A S H E E T

4 Biomarker-Specific Antibodies Alpha fetoprotein Haptoglobulin MMP-2 Alpha1 antichymotrypsin Hemoglobin MMP-3 Alpha 2 macroglobulin Hepatocyte growth factor MMP-9 Angiogenin ICAM-1 Myeloperoxidase Angiopoietin-2 IgA Myoglobin Angiostatin IgG Neuron-specific enolase Apolipoprotein A1 IgM RANTES Apolipoprotein J IL-1- Osteopontin Beta2 microglobulin IL1- PDGF (all isoforms) Bone sialoprotein IL-2 PDGF (BB isoform only) CA15-3 IL-2 receptor- Placental alkaline phosphatase CA19-9 IL-2 receptor- Plasminogen CA 50 IL-3 Plasminogen activator inhibitor CA125 IL-4 Prostatic acid phosphatase Carcinoembryonic antigen (group 2 specific) IL-5 PSA (free) Carcinoembryonic antigen (group 4 specific) IL-6 PSA (total) Cathepsin B IL-7 PSA-ACT complex Ceruloplasmin IL-8 S100 Chondroitin sulftate IL-10 Serum albumin Chorionic gonadotropin- IL-12p40 Sialyl Lewis X Chorionic gonadotropin- IL12-p70 TAG-72 Chromogranin IL-13 Tetranectin Collagen type I IL-17 TGF- Complement C4 Insulin TGF- C-Reactive protein Insulin growth factor binding protein 3 Thrombospondin-1 Cyclin-dependent kinase inhibitor 2A Insulin-like growth factor 1 Thrombopoietin Cytokeratin fragment 21-1 (CYFRA 21-1) Interferon- Thyroglobulin Eotaxin IP-10 TIMP1 Epidermal growth factor Kallikrein-5 TIMP2 Epidermal growth factor receptor Kallikrein-9 TNF- ErbB2 Kallikrein-12 TNF- E-selectin Kallikrein-14 Transferrin Estrogen receptor Laminin Tumor-associated trypsin inhibitor Fas Low-density lipoprotein Tyrosinase Fas ligand MCP-1 Urokinase plasminogen activator Ferritin MCP-2 VCAM-1 Fibroblast growth factor-7 MCP-3 VE-cadherin Fibroblast growth factor-basic MCP-4 VEGF G-CSF M-CSF VEGF-D GM-CSF MIP-1- Von Willebrand factor Sample Processing Service Whatman recognizes that not all scientists have access to the software and instrumentation needed to process and scan microarrays, and to collect and analyze the resulting data. The Whatman serum biomarker sample processing service makes it simple for researchers to outsource the tests or try this new technology before adopting it in their labs. Customer samples are sent to Whatman and processed using the Serum Biomarker Chip Data are uploaded to a passwordprotected ftp site within 10 business days of receipt of samples at Whatman Custom Serum Biomarker Chips Add up to 48 additional antibodies to the existing Serum Biomarker Chip array Additional antibodies are either provided by the customer or customer-specified commercial antibodies Chips are shipped with an antibody map Labeling and detection reagents are available in bulk quantities (see ordering information) Contact Whatman customer service to discuss your requirements W H A T M A N S E R U M B I O M A R K E R C H I P D A T A S H E E T

5 One standard Serum Biomarker Kit Contains 2 slides Each slide contains 2 arrays for use with 1 serum sample pair One Two-Color Labeling and Fluorescent Detection Kit contains sufficient reagents to label the two slides in a standard Serum Biomarker Chip Kit. Serum Biomarker Chips and two-color labeling and detection reagents may be purchased in bulk quantities: 24 slides (for 24 paired samples or 48 total samples) 96 slides (for 96 paired samples or 192 total samples) Ordering Information Description Qty/Pkg Item # Serum Biomarker Chip The kit contains: Serum Biomarker Chip Arrayed FAST Slides 2 Dual Pad Incubation/Processing Chambers 2 Protein Array Wash Buffer 1 x 125 ml Protein Array Blocking Buffer 1 x 5 ml Bulk Serum Biomarker Chip Kits 24 chips (contain the same items as the 2-chip kit) 96 chips Fast Frame Slide Holder for 4 slides Chip Clip Slide Holder for 1 slide Two-color Labeling & Fluorescent Detection Kit The kit contains: Biotin-ULS 20 µl Fluorescein-ULS 20 µl 10x Protein Labeling Buffer 80 µl 10x KREAstop 80 µl Streptavidin-DY647 Conjugate 150 µl Anti-Fluorescein Antibody-DY 547 conjugate 350 µl Micro Bio-Spin Chromatography Columns 8 User Manual 1 Bulk Two-Color Labeling & Fluorescent Detection Reagents Two-Color Labeling Kit 24 (for 24 chips) Two-Color Labeling Kit 96 (for 96 chips) ArrayVision FAST Software Single User Software ArrayVision Demo USA/Canada ArrayVision FAST Software Compatible with virtually all image formats used to analyze fluorescent microarrays. Comprehensive normalization and background correction includes Dy 547/Dy 647 functions to improve accuracy in comparing protein abundance. Scatter plot and elemental display provide quick data assessment and reports based on user-defined criteria. Template definitions and analysis parameters are contained in the Serum Biomarker Chip-defined protocol. Data analysis is performed using the SBC Analysis Workbook, a preconfigured Microsoft Excel file that converts fluorescent data into Dy547/Dy647 ratio values; available at Seamless data export to Microsoft Access, XML, GeneSpring and DecisionSite. Chip Clip The BioScience Chip Clip securely holds the FAST Slide 2-pad slide and incubation chamber for processing of the Serum Biomarker Chip. The Chip Clip, used in conjunction with dual-well silicone Incubation Chambers, ensures leak-proof barriers around the two arrayed pads on the Serum Biomarker Chip Slide. The slide and Incubation Chamber are easily inserted into and removed from the Chip Clip; side rails hold the chamber firmly against the slide surface. Manufactured from durable, injection-molded Delrin. SBC Analysis Workbook Downloadable at For the latest protocols and product information, visit The Whatman Serum Biomarker Chip is intended for research purposes only, not for diagnostic use. W H A T M A N S E R U M B I O M A R K E R C H I P D A T A S H E E T

6 Whatman Quality Whatman is a global leader in separations technology and is known in the scientific community for providing Innovative Life Science products and solutions. Our instinct for simplification accelerates the rate of discovery, reduces costs and saves time. For more information, visit Whatman, FAST and Chip Clip are trademarks of the Whatman Group. ULS is licensed from and a registered trademark of KREATECH Biotechnology B.V., and covered by one or more patents of KREATECH Biotechnology BV, including, but not restricted to, the following: EP 0,539,466; US 5,580,990; US 5,714,327; US 5,985,566; US 6, ; and EP 1,019,420. DY 647 and DY 547 are registered trademarks of Dyomics GmbH. DY 647 is covered by the license of Dyomics GmbH, Germany, to produce and distribute dyes for in vitro applications claimed by the patent family DE Delrin is a registered trademark of E.I. dupont de Nemours and Company. ArrayVision is a trademark of GE Healthcare. Bio-Spin is a registered trademark of Bio-Rad Laboratories, Inc. GeneSpring is a trademark of Silicon Genetics. DecisionSite is a trademark of Spotfire, Inc. Microsoft, Access and Excel are either registered trademarks or trademarks of Microsoft Corporation in the United States and/or other countries. North America Europe Whatman Inc. 200 Park Avenue, Suite 210 Florham Park, NJ USA Tel: WHATMAN (US and Canada) Fax: info@whatman.com Whatman International Ltd. Springfield Mill, James Whatman Way Maidstone Kent ME14 2LE UK Tel: +44 (0) Fax: +44 (0) information@whatman.com Whatman GmbH Hahnestrasse 3 D Dassel Germany Tel: +49 (0) Fax: +49 (0) information@whatman.com Japan Asia Pacific Whatman Japan KK JPR Ichigaya Building 6F Kudan-Minami, Chiyoda-ku, Tokyo Japan Tel: +81 (0) Fax: +81 (0) japaninfo@whatman.com Whatman Asia Pacific Pte Ltd. 171 Chin Swee Road #08-01 San Centre Singapore Tel: Fax: wap@whatman.com (US) S (EU) 04/06