Bio-Rad Bio-Plex Pro Human Cancer Biomarker Assays User Manual
Bio-Plex Pro
™
Human
Cancer Biomarker Assays
Instruction Manual
For technical support, call your local Bio-Rad office, or in the U.S., call 1-800-424-6723.
For research use only. Not for diagnostic procedures.
Table of Contents
Introduction 1
Principle 2
Kit Contents and Storage 4
Recommended Materials 5
Assay Workflow 6
lmportant Considerations 7
Detailed Instructions 7
1. Plan Plate Layout 8
2. Prepare Instrument 9
3. Prepare Wash Method 10
4. Prepare Standards and Controls 11
5. Prepare Samples 13
6. Prepare Coupled Beads 16
7. Run Assay 17
8. Read Plate 21
Troubleshooting Guide 29
Plate Layout Template 34
Calculation Worksheet 35
Safety Considerations 37
Legal Notices 37
Ordering Information 38
Introduction
Bio-Plex Pro™ Human Cancer Biomarker Assays
Bio-Plex Pro human cancer biomarker assays are designed to meet
the needs of the most demanding preclinical and clinical research
environments. These magnetic bead–based multiplex immunoassays
include a unique blend of soluble biomarkers involved in disease
processes such as angiogenesis, metastasis, inflammation, cell
proliferation, cell adhesion, and apoptosis. With diluents optimized
for enhanced performance in clinical matrices, a fast protocol, and a
flexible kit format, the human cancer biomarker assays promise to make
multiplexing easier and more reliable than ever.
Multiplexing with Bio-Plex Pro Assays
Bio-Plex Pro assays enable researchers to quantify multiple protein
biomarkers in a single well of a 96-well plate in just three to four hours.
These robust immunoassays require as little as 12.5 µl of serum or
plasma or 50 µl of other biological fluid. The use of magnetic (MagPlex)
beads allows researchers to automate wash steps on a Bio-Plex Pro (or
similar) wash station. Magnetic separation offers greater convenience and
reproducibility compared to vacuum filtration.
For more information please visit www.bio-rad.com/bio-plex.
1
Principle
Technology
The Bio-Plex
®
suspension array system is built upon the three core
elements of xMAP technology:
n
Fluorescently dyed microspheres (also called beads), each with a distinct
color code or spectral address to permit discrimination of individual tests
within a multiplex suspension. This allows simultaneous detection of more
than 100 different types of molecules in a single well of a 96-well microplate
n
A dedicated flow cytometer with two lasers and associated optics to
measure the different molecules bound to the surface of the beads
n
A high-speed digital signal processor that efficiently manages the
fluorescence data
Assay Format
Bio-Plex Pro
™
human cancer assays are essentially immunoassays
formatted on magnetic beads. The assay principle is similar to that of
a sandwich ELISA (Figure 1). Capture antibodies directed against the
desired biomarker are covalently coupled to the beads. Coupled beads
react with the sample containing the biomarker of interest. After a series
of washes to remove unbound protein, a biotinylated detection antibody
is added to create a sandwich complex. The final detection complex is
formed with the addition of streptavidin-phycoerythrin (SA-PE) conjugate.
Phycoerythrin serves as a fluorescent indicator, or reporter.
2
Biomarker
of Interest
Streptavidin
Magnetic Bead
Capture
Antibody
Fig. 1. Bio-Plex sandwich immunoassay.
Biotinylated
Detection
Antibody
Phycoerythrin
Fluorescent
Reporter
Data Acquisition and Analysis
Data from the reactions are acquired using a Bio-Plex system or similar
Luminex-based reader. When a multiplex assay suspension is drawn into
the Bio-Plex 200 reader, for example, a red (635 nm) laser illuminates
the fluorescent dyes within each bead to provide bead classification
and thus assay identification. At the same time, a green (532 nm)
laser excites PE to generate a reporter signal, which is detected by a
photomultiplier tube (PMT). A high-speed digital processor manages
data output, and Bio-Plex Manager
™
software presents data as median
fluorescence intensity (MFI) as well as concentration (pg/ml). The
concentration of analyte bound to each bead is proportional to the MFI
of the reporter signal.
3
Kit Contents and Storage
Reagents Supplied
Bio-Plex Pro
™
human cancer biomarker assays are available in a
convenient all-in-one kit format that includes assay, reagent, and diluent
components in a single box.
Table 1. Contents of 1 x 96-well premixed and custom x-Plex™ kits.
**
Component
Standard diluent HB
Sample diluent HB
Assay buffer
Wash buffer
Detection antibody diluent
Streptavidin-PE (100x)
Assay plate (96-well flat bottom plate or filter plate)
Sealing tape
Assay Quick Guide
Coupled magnetic beads (20x)
Detection antibodies (20x)
Standard
Quality Control High*
Quality Control Low
* Only premixed cancer panels and custom cancer x-Plex™ kits include quality controls.
** Volumes shown are approximate. Quantities in Express assays will vary.
*
Quantity
1 bottle (10 ml)
1 bottle (8 ml)
1 bottle (50 ml)
1 bottle (200 ml)
1 bottle (5 ml)
1 tube
1 plate
1 pack of 4
1 booklet
1 tube
1 tube
1 vial
1 vial
1 vial
Storage and Stability
Kit contents should be stored at 4°C and never frozen. Coupled magnetic
beads and streptavidin-PE should be stored in the dark. All components
are guaranteed for a minimum of six months from the date of purchase
when stored as specified.
4
Table 2. Recommended materials.
Item
Bio-Plex Pro Assays Quick Guide 1
Ordering Information
Bulletin #100-22581 (download
at www.bio-rad.com/bio-plex)
®
Bio-Plex
Bio-Plex validation kit
200 system or Luminex system with HTF
Bio-Rad catalog #171-000205
Bio-Rad catalog #171-203001
Note: Run the validation kit monthly to ensure optimal
performance of fluidics and optics systems
Bio-Plex calibration kit
Bio-Rad catalog #171-203060
Note: Run the calibration kit daily to standardize
fluorescence signal
Bio-Plex Pro wash station
Bio-Rad catalog #300-34376
For use with magnetic bead-based assays only
Bio-Plex Pro II wash station
Bio-Rad catalog #300-34377
For use with both polystyrene (nonmagnetic) and magnetic
bead-based assays
Bio-Plex handheld magnetic washer
Bio-Rad catalog #170-20100
For use with magnetic bead–based assays only
Bio-Plex Pro flat bottom plates (forty 96-well plates)
Bio-Rad catalog #171-025001
For magnetic separation on the Bio-Plex Pro wash station
Microtiter plate shaker
IKA MTS 2/4 shaker for 2 or 4 microplates
IKA catalog #320-8000
or
Barnstead/Lab-Line Model 4625 plate
VWR catalog #57019-600
shaker (or equivalent capable of 300–1,100 rpm)
®
Bio-Rad
Aurum™ vacuum manifold
Bio-Rad catalog #732-6470
For vacuum filtration
BR-2000 vortexer
Reagent reservoirs, 25 ml
For capture beads and detection antibodies
Reagent reservoir, 50 ml (for reagents and buffers)
Pall Life Science Acrodisc: 25 mm PF syringe filter
(0.8/0.2 µm Supor membrane)
Filter plate, 1 x 96 with clear plastic lid and tray
Bio-Rad catalog #166-0610
VistaLab catalog #3054-1002
or
VistaLab catalog #3054-1004
VistaLab catalog #3054-1006
Pall Life Sciences
catalog #4187
Bio-Rad catalog #171-304502
Other: 15 ml polypropylene tubes for reagent dilutions, calibrated pipets, pipet tips, sterile
distilled water, aluminum foil, absorbent paper towels, 1.5 or 2 ml microcentrifuge tubes, and
standard flat bottom microplate (for calibrating vacuum manifold).
5
Assay Workflow
Prewet wells
(for lter plate only)
Add 50 μl 1x beads to wells
Wash 2 x 100 μl
Add 50 μl standards, samples, controls,
incubate 1 hr at RT with shaking at 850 rpm
Wash 3 x 100 μl
Add 25 μl 1x detection antibody, incubate
30 min at RT with shaking at 850 rpm
Wash 3 x 100 μl
Add 50 μl 1x streptavidin-PE, incubate
10 min at RT with shaking at 850 rpm
Wash 3 x 100 μl
Resuspend in 125 μl assay buffer,
shake at 850 rpm for 30 sec
Acquire data on Bio-Plex system
6
lmportant Considerations
Instruments and Software
The Bio-Plex Pro
™
assays described in this manual are compatible with
all currently available Luminex-based life science research instruments.
Assays can be read and analyzed with either Bio-Plex Manager
™
software
or Luminex xPONENT software (section 8).
Assay Procedures
Please pay close attention to vortexing, shaking, and incubation times
and to Bio-Plex
®
reader PMT (RP1) setting, as these have been optimized
specifically for each assay panel.
Assay Quick Guide
Each assay kit comes complete with a printed Bio-Plex Pro
™
Assay Quick Guide
(bulletin #10022581), which can be used to prepare and run a full 1 x 96-well
assay plate. Users can also download a copy at www.bio-rad.com/bio-plex.
Bead Regions and Multiplexing Compatibility
n
Bead regions for all analytes are listed in the Read Plate section.
n
It is not recommended to mix analytes between the different
cancer panels, or with other Bio-Plex assay panels or reagent kits.
For example:
a. Receptors should not be mixed with their ligands
b. Overlapping analytes in different antigen standards should not be mixed
c. Bio-Plex Pro reagent kit II contains diluents that have not been fully
validated in other assays
In each case, standard curves and sample values may be inaccurate.
Detailed Instructions
The following pages provide detailed instructions for each step of the
assay procedure, including preparation, running the assay, and reading the
plate with Bio-Plex Manager
7
™
and Luminex xPONENT software.
1. Plan Plate Layout
Prior to running the assay, determine the total number of wells in the
experiment using the Plate Layout Template on page 34 or the Plate
Formatting tab in Bio-Plex Manager
in Figure 2, with all conditions in duplicate.
1. Assign standards to columns 1 and 2, with the highest
concentration in row A and the lowest concentration in row H.
2. Assign the blank to wells A3 and A4. The blank should consist of your
chosen standard diluent. Note that Bio-Plex Manager automatically
subtracts the blank (B) MFI value from all other assay wells.
3. User-specified controls, as well as the quality controls supplied in
premixed cancer biomarker kits, are assigned to wells in columns 3 and 4.
4. The remainder of the plate is available for samples.
5. Once the total number of wells is known, you can calculate the
required volumes of beads, detection antibody, and streptavidin-PE.
Use Tables 6, 9, and 11, respectively, or the Calculation Worksheet on
pages 35–36.
Fig. 2. Suggested plate layout. For detailed instructions on
plate formatting in Bio-Plex Manager, see section Read Plate. Legend
S Standard
™
. A suggested plate layout is shown
B Blank
X Samples
C Controls
8
2. Prepare Instrument
Start up and calibrate the Bio-Plex® system with Bio-Plex Manager™
software prior to setting up the assay. The calibration kit should be run
daily or before each use of the instrument to standardize the fluorescent
signal. For instructions on using other xMAP system software packages,
contact Bio-Rad Technical Support.
The validation kit should be run monthly to ensure optimal performance of
fluidics and optics systems. Refer to either the software manual or online
Help for directions on how to conduct validation.
Start Up System (Bio-Plex 100, 200, or similar)
1. Empty the waste bottle and fill the sheath fluid bottle before starting
if high throughput fluidics (HTF) are not present. This will prevent
fluidic system backup and potential data loss.
2. Turn on the reader, XY platform, and HTF (if included). Allow the
system to warm up for 30 min (if not already done).
3. Select Start up and follow the instructions. If the system is idle
for 4 hr without acquiring data, the lasers will automatically turn off.
To reset the 4-hr countdown, select Warm up and wait for the
lasers/optics to reach operational temperature.
Calibrate System
1. Select Calibrate and confirm that the default values for CAL1
and CAL2 are the same as the values printed on the bottle of
Bio-Plex calibration beads. Use the Bio-Plex system low RP1
target value.
2. Select OK and follow the software prompts for step-by-step
instructions for CAL1 and CAL2 calibration.
Note! In Bio-Plex Manager version 6.1 and higher, startup, warm up,
and calibration can be performed together by selecting the “Start up and
calibrate” icon.
9
3. Prepare Wash Method
Bio-Plex Pro assays are compatible with both magnetic separation and
vacuum filtration methods. However, for best results, we recommend
performing the assays in a flat bottom plate with magnetic separation.
Table 3. Summary of compatible wash stations and plate types.
Wash Method Wash Station Assay Plate
Magnetic separation Bio-Plex Pro
Bio-Plex Pro II (use MAG programs)
Bio-Plex handheld magnetic washer
Vacuum filtration Bio-Plex Pro II (use VAC programs) Filter plate
Vacuum manifold (manual)
Setting up the Bio-Plex Pro or Bio-Plex Pro II
Wash Station
The wash station does not require calibration; however, it should be primed
before use. For more information, refer to the Bio-Plex Pro and Pro II wash
station quick guide (bulletin #5826).
1. Install the appropriate plate carrier on the wash station.
2. Use the Prime procedure to prime channel 1 with wash buffer.
Setting up the Bio-Plex Handheld Magnetic Washer
Place an empty flat bottom plate on the magnetic washer by sliding it
under the retaining clips. Push the clips inward to secure the plate. Make
sure the plate is held securely. If needed, the clips can be adjusted for
height and tension. For detailed instructions, refer to the user guide
(bulletin #10023087).
™
Flat bottom plate
Setting up a Vacuum Manifold
Calibrate the vacuum manifold by placing a standard 96-well flat bottom
plate on the unit and adjusting the pressure to –1 to –3" Hg. In general,
100 µl liquid should take 3–4 sec to clear the well. For more detailed
instructions, refer to bulletin #10005042.
10
4. Prepare Standards and Controls
General Instructions
n
It is essential to prepare standards and quality controls (if included)
exactly as described in this section. Incorrect preparation may lead to
low signal or variable measurements from plate to plate
n
The product data sheet or peel-off sticker provided with the standards
lists the most concentrated point on the standard curve (S1). Enter this
information into Bio-Plex Manager
Using the Quality Controls (optional)
Two-level quality controls (high and low) are included with premixed
cancer biomarker panels and custom x-Plex
for monitoring the day-to-day quality of assay results.
Selecting a Diluent for Standards and Controls
Refer to Table 4 for recommended diluents based on different sample types.
™
software as instructed in section 8
™
kits. Their use is intended
In order to meet the lot-specific control ranges provided on the product data
sheet, both the standards and controls should be reconstituted in Bio-Plex
®
standard diluent HB. If reconstituting in a different diluent, users will need to
establish/validate their own control ranges or acceptance criteria.
Table 4. Summary of recommended diluents for standards and controls.
Diluent for Standards
Sample Type and Controls* Add BSA
Serum and plasma Standard diluent HB None
Culture media, with serum Culture medium None
Culture media, serum-free Culture medium To 0.5% final**
Lavage, sputum, other fluids Sample diluent HB To 0.5% final**
Lysate Sample diluent HB To 0.5% final**
* If using diluents other than standard diluent HB, users must establish their own control ranges.
** At least 0.5% final BSA is recommended to stabilize analytes and reduce adsorption to labware.
11
Loading...