Serum Biomarker Chip Datasheet - Whatman

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

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)
Labeling and Detection Procedure Overview
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
Sample A Sample B Sample A Sample B
Block slide with
S&S Blocking Buffer
for 30 min
at room
temperature
Mix 1
Mix 2
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.
Label serum proteins with
Biotin-ULS ( ) and
Fluorescein-ULS ( )
for 2-6 hours at 42 °C
Terminate reaction
and remove unbound
ULS reagent
with spin columns
Pool Samples
Incubate labeled proteins
with Serum Biomarker Chip
for 4 hours to overnight at room temperature
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
Wash 6X to remove
unbound proteins

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-Dy647 and anti-fluorescein
antibody-Dy547 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 0.
Incubate Serum Biomarker Chip
with two-color fluorescent
detection solution
for 1 hour at room temperature
Log2 Ratio
Log2 Ratio
Log2 Ratio
Log2 Ratio
0.40
0.30
0.20
0.10
0.00
-0.10
-0.20
-0.30
-0.40
-0.50
2.00
1.50
1.00
0.50
0.00
-0.50
-1.00
-1.50
1.50
1.00
0.50
0.00
-0.50
-1.00
-1.50
-2.00
0.50
0.40
0.30
0.20
0.10
0.00
-0.10
-0.20
Wash 6X to remove
unbound proteins
Excite
547 nm
Bladder Cancer
Breast Cancer
Colon Cancer
Prostate Cancer
Scan at two wavelengths,
spot-find, and sample data
Excite
647 nm
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
Log2 Ratio
0.50
0.40
0.30
0.20
0.10
0.00
-0.10
-0.20
Prostate Cancer
Bioinformatic
analysis

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
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
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

One standard Serum
Biomarker Kit
Contains
2 slides
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
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.
Description Qty/Pkg Item #
Serum Biomarker Chip 10 486 077
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 10 486 098
(contain the same items as the 2-chip kit) 96 chips 10 486 099
Fast Frame Slide Holder for 4 slides 1 10 486 001
Chip Clip Slide Holder for 1 slide 1 10 486 081
Two-color Labeling & Fluorescent Detection Kit 10 486 085
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-DY547 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) 10 486 100
Two-Color Labeling Kit 96 (for 96 chips) 10 486 101
ArrayVision FAST Software
Single User Software 10 486 035
ArrayVision Demo USA/Canada 10 486 034
SBC Analysis Workbook Downloadable at www.arraying.com
For the latest protocols and product information, visit www.arraying.com.
The Whatman Serum Biomarker Chip is intended for research purposes only, not for diagnostic use.
ArrayVision
FAST Software
• Compatible with virtually all image
formats used to analyze fluorescent
microarrays.
• Comprehensive normalization and
background correction includes
Dy547/Dy647 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 www.arraying.com.
• Seamless data export to Microsoft ®
Access, XML, GeneSpring and
DecisionSite.
Chip Clip
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
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 ® .

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 www.whatman.com
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 6,133.038; and EP 1,019,420.
DY647 and DY547 are registered trademarks of Dyomics GmbH. DY647 is covered by the
license of Dyomics GmbH, Germany, to produce and distribute dyes for in vitro applications
claimed by the patent family DE 44 45 065. 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.
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