Bio-Plex Pro™ Human - Bio-Rad
Bio-Plex Pro Human
™
Th17 Cytokine 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
Introduction1
Principle2
Kit Contents and Storage
4
Recommended Materials
5
Assay Workflow
6
Important Considerations
7
Detailed Instructions
1. Plan Plate Layout
7
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
22
Troubleshooting Guide 29
Plate Layout Template
34
Calculation Worksheet
35
Safety Considerations
37
Legal Notices
37
Ordering Information
38
Introduction
Bio-Plex Pro™ Human Th17 Cytokine Assays
Bio-Plex Pro human Th17 cytokine assays are designed to meet the needs
of the most demanding preclinical and clinical research environments.
These magnetic bead-based multiplex immunoassays include a
comprehensive blend of soluble biomarkers involved in the T-helper cell
type 17 immune response pathway, a key driver of autoimmune diseases,
as well as the body’s response to microbial infection. With diluents
optimized for enhanced performance in clinical matrices, a fast protocol,
and a flexible kit format, the Human Th17 cytokine 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 3 to 4 hr. 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 around the three core
elements of xMAP technology:
• 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
• A dedicated flow cytometer with two lasers and associated optics to
measure the different molecules bound to the surface of the beads
• A high-speed digital signal processor that efficiently manages the fluorescence data
Assay Format
Bio-Plex Pro™ 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
BIOTINYLATED
DETECTION
ANTIBODY
PHYCOERYTHRIN
FLUORESCENT
REPORTER
Figure 1. Bio-Plex sandwich immunoassay
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 reporter signal.
3
Kit Contents and Storage
Reagents Supplied
Bio-Plex Pro™ human Th17 cytokine 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
Quantity**
10 ml
Sample diluent HB
8 ml
Assay buffer
50 ml
Wash buffer
200 ml
Detection antibody diluent
Streptavidin-PE (100x)
Assay plate (96-well flat-bottom plate or filter plate)
Sealing tape
Assay Quick Guide
5 ml
1 tube
1 plate
1 pack of 4
1 booklet
Coupled magnetic beads (20x)
1 tube
Detection antibodies (20x)
1 tube
Standard
1 vial
Quality Control High*
Quality Control Low*
1 vial
1 vial
* Only premixed Th17 panels and custom Th17 x-Plex kits include quality controls.
** Volumes shown are approximate. Quantities in Express assays will vary.
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 6 months from the date of purchase
when stored as specified.
4
Table 2. Recommended materials.
Item
Ordering Information
Bio-Plex Pro Assays Quick Guide 1
Item #10022581 (download at
www.bio-rad.com/bio-plex)
Bio-Plex® 200 system or Luminex system with HTF
Bio-Rad catalog #171-000205
Bio-Plex validation kit
Note: the validation kit should be run monthly to ensure
performance of fluidics and optics systems
Bio-Rad catalog #171-203001
Bio-Plex calibration kit
Note: the calibration kit should be run either daily or
whenever the instrument is to be used to standardize
fluorescence signal
Bio-Rad catalog #171-203060
Bio-Plex Pro wash station
For use with magnetic bead-based assays only
Bio-Rad catalog #300-34376
Bio-Plex Pro II wash station
For use with both polystyrene (nonmagnetic) and magnetic
bead-based assays
Bio-Rad catalog #300-34377
Bio-Plex handheld magnetic washer
For use with magnetic bead-based assays only
Bio-Rad catalog #170-20100
Bio-Plex Pro flat-bottom plates (40, 96-well plates)
For magnetic separation on the Bio-Plex Pro wash station
Bio-Rad catalog #171-025001
Microtiter plate shaker
IKA MTS 2/4 shaker for 2 or 4 microplates
or
Barnstead/Lab-Line Model 4625 plate
shaker (or equivalent capable of 300–1,100 rpm)
IKA catalog #320-8000
VWR catalog #57019-600
Bio-Rad Aurum™ vacuum manifold
For vacuum filtration
Bio-Rad catalog #732-6470
BR-2000 vortexer
Bio-Rad catalog #166-0610
Reagent reservoirs, 25 ml
For capture beads and detection antibodies
VistaLab catalog #3054-1002
or
VistaLab catalog #3054-1004
Reagent reservoir, 50 ml (for reagents and buffers)
VistaLab catalog #3054-1006
Pall Life Science Acrodisc: 25 mm PF syringe filter
(0.8/0.2 µm Supor membrane)
Pall Life Sciences
catalog #4187
Filter plate, 1 x 96 with clear plastic lid and tray
Bio-Rad catalog #171-304502
Other: 15 ml polypropylene tubes for reagent dilutions. Calibrated pipets (multichannel pipets
are optimal for this application) and 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 filter plate only)
Add beads
Wash 2x
Add standards, samples, controls, 1 hr
Wash 3x
Add detection antibody, 30 min
Wash 3x
Add streptavidin-PE, 10 min
Wash 3x
Resuspend
Acquire data
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 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 (item #1002258), 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 B
ead regions for all analytes are listed in the Read Plate section.
n
F
or optimum performance of the human Th17 cytokine assays, mixing
of Th17 analytes with other Bio-Plex assay panels or reagent kits is not
recommended. For example:
a. Overlapping analytes in different antigen standards should not be mixed
b. 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™ and Luminex xPonent software.
7
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™. A suggested plate layout is shown
in Figure 2, with all conditions run 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) FI value from all other assay wells.
3.User-specified controls, as well as the quality controls supplied in
premixed Th17 cytokine kits, are assigned to wells in colums 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 8.Legend
S
Standards
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 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 v6.1 and higher, startup, warm up and calibrate
can be performed together by selecting the start up and calibrate icon.
9
3. Prepare Wash Method
There are two methods of performing wash steps in the assay procedure:
magnetic separation and vacuum filtration. Compatible wash stations and
plate types are shown in the table below.
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
Flat-bottom plate
Vacuum filtration
Bio-Plex Pro II (use VAC programs)
Vacuum manifold (manual)
Filter plate
Magnetic Separation Using 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.
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
(item #10023087).
Vacuum Filtration Using 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 ul liquid should take 3–4 sec to clear the well. For more detailed
instructions, refer to item #10005042.
10
4. Prepare Standards and Controls
General Instructions
• 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
• 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™ software as instructed in Section 8
Using the Quality Controls (optional)
Two-level quality controls (high and low) are included with premixed
Th17 cytokine panels and custom x-Plex kits. Their use is intended 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.
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 absorption to labware.
11
Reconstitute Standards and Quality Controls
This procedure prepares enough standard to run each dilution in duplicate.
1. Gently tap the vial containing the lyophilized standards on a solid
surface to ensure the pellet is at the bottom of the vial.
2.Reconstitute a single vial of standards with 781 µl of the appropriate
diluent. Optional: at the same time, reconstitue the two control vials
with 250 µl of the appropriate diluent as summarized in Table 4.
Controls do not require further dilution.
3. Gently vortex the reconstituted standards and controls for 5 sec,
then incubate on ice for 30 min. It is important that reconstitution
of standards and controls is started and ended at the same time.
Be consistent with this incubation time to ensure optimal assay
performance and reproducibility.
4. During the incubation period, prepare the samples as instructed in the
Prepare Samples section.
Prepare the Standard Dilution Series
The following procedure produces an eight-point standard curve with a
four-fold dilution between each point. Pipet carefully using calibrated pipets
and use new pipet tips for every volume transfer.
1. Label eight 1.5 ml polypropylene tubes S2 through S8 and Blank.
2. Add 150 µl of the appropriate diluent to tubes S2–S8 (Figure 3).
3. V
ortex reconstituted standards gently for 5 sec before removing
any volume. Add 50 µl to the S2 tube containing the chosen standard
diluent. Vortex for 5 sec.
12
4. Use a new pipet tip to transfer 50 µl from the S2 tube to the
S3 tube. Vortex for 5 sec.
5. Continue with 1:4 (fourfold) serial dilutions as shown in Figure 3.
6. Use reconstituted and diluted standards and controls immediately. Do
not freeze for future use.
Fig. 3. Preparing a fourfold dilution series with a single reconstituted standard.
50
Reconstituted
Standard
S1
50 505050 5050
150 150 150 150150 150150150
Transfer Volume, µl
Diluent, µl
S2 S3S4S5S6S7S8
Blank
5. Prepare Samples
General guidelines for preparing different sample types are provided
here. For more information, consult publications listed in Bio-Rad bulletin
5297, available for download at www.bio-rad.com, or contact Bio-Rad
Technical Support.
• Once thawed, keep samples on ice. Prepare dilutions just prior to the
start of the assay and equilibrate to room temperature before use
• Do not freeze diluted samples.
13
Table 5. Summary of recommended sample diluents and dilution factors.
Sample Type
Diluent
Add BSA
Sample Dilution*
Serum and plasma
Sample diluent HB
None
Fourfold (1:4)
Culture media, with serum
Culture media
None
User optimized (neat to 1:10)
Culture media, serum-free
Culture media
To 0.5% final*
User optimized (neat to 1:10)
Lavage, lysate, other fluids
Sample diluent HB
To 0.5% final*
User optimized (neat to 1:10)
Lysate
Sample diluent HB
To 0.5% final*
User optimized (at least 1:2 for
50 to 500 ug/ml final protein)
*At least 0.5% final BSA is recommended to stabilize analytes and reduce absorption to labware.
Sample Preparation
Serum and Plasma
EDTA or citrate is preferred as an anticoagulant. Heparin-treated plasma,
while compatible with Bio-Plex Pro™ assays, may absorb certain soluble
proteins of interest. Avoid using hemolyzed samples as this may lead to
false positive results.
1. Draw whole blood into collection tubes containing anticoagulant. Invert tubes several times to mix.
2. For serum, allow blood to clot at room temperature for 30 to 45 min.
For plasma, proceed directly to the centrifugation steps.
3. Perform centrifugation at 1,000 x g for 15 min at 4°C and transfer the
serum or plasma to a clean polypropylene tube.
4. To completely remove platelets and precipitates, centrifuge again at 10,000 x g for 10 min at 4°C.
5.Dilute samples fourfold (1:4) by adding 1 volume of sample to 3
volumes of Bio-Plex® sample diluent HB (for example, 40 µl sample +
120 µl sample diluent).
6. Assay samples immediately or aliquot into single-use tubes and store at –70°C. Avoid repeated freeze-thaw cycles.
14
Cell Culture Supernatant
1. Collect supernatants and centrifuge at 1,000 x g for 15 min at 4°C.
For cell lines cultured in serum-free culture media, collect
samples and add BSA as a carrier protein to a final concentration
of at least 0.5% to stabilize protein analytes and to prevent
adsorption to labware.
2. Transfer to a clean polypropylene tube. If cellular debris or precipitates are present, centrifuge again at 10,000 x g for 10 min at 4°C.
3.We recommend testing undiluted samples first. If levels are
anticipated to be high, samples can be further diluted in culture
medium. Rarely would samples need to be diluted greater than 1:10.
4. Assay immediately or store samples in single-use aliquots at –70°C.
Avoid repeated freeze-thaw cycles.
Lavage, Sputum, and Other Biological Fluid Samples
Keep all samples on ice until ready for use. The appropriate sample
dilution factor should be optimized by the user.
1.If required, dilute the sample in Bio-Plex sample diluent with BSA
added to a final concentration of 0.5%.
2.Centrifugation at 10,000 x g for 10 min at 4°C may be required to
clarify the sample.
Lysates
The Bio-Plex cell lysis kit is required for lysate preparation (available
separately, catalog #171-304011 and #171-304012). Refer to bulletin 5297
for a list of published articles on cytokine analysis in tissue samples.
1.Prepare the cell or tissue lysates according to the instructions provided
with the Bio-Plex cell lysis kit. The protease inhibitors, Factor I and II,
are included in the kit. PMSF needs to be added to lysis buffer at a final
concentration of 2 mM. The lysates should be free of particulate matter.
15
2.Determine the total protein concentration of the lysate. It may be
necessary to test lyse your samples with different volumes of lysing
solution to obtain the specified protein concentration range.
3.Dilute at least 1:2 in sample diluent + 0.5% BSA, to a final protein
concentration of 50–500 µg/ml.
For optimum antibody binding during sample incubation, it is
important to dilute lysates as much as possible to reduce the
detergent concentration.
4. If the lysate is not tested immediately, store at –20°C. Lysates are
stable for up to five freeze-thaw cycles.
6. Prepare Coupled Beads
1.Use Tables 6–7 or the Calculation Worksheet on page 35 to calculate
the volume of coupled beads and assay buffer needed. Tables 6–7
include ~20% excess to compensate for transfer loss.
2. Add the required volume of Bio-Plex® assay buffer to a 15 ml
polypropylene tube.
3. Vortex the 20x stock of coupled beads at mid speed for 30 sec.
Carefully open the cap and pipet any liquid trapped in the cap back
into the tube. This is important to ensure maximum bead recovery.
Do not centrifuge the vial; doing so will cause the beads to pellet.
4.Dilute coupled beads to 1x by pipetting the required volume into the
15 ml tube. Vortex.
Each well of the assay requires 2.5 μl of the 20x stock adjusted to a
final volume of 50 μl in assay buffer.
5. Protect the beads from light with aluminum foil. Equilibrate to room
temperature prior to use.
16
Tables 6–7 summarize the volumes required to prepare 1x beads from
a 20x stock. Also shown are volumes for preparing 1x beads when mixing
two 20x stocks of single-plex assays.
Preparing 1x coupled beads from 20x stock
Table 6. Premixed panel or one singleplex assay.
# of Wells
20x Beads, µl Assay Buffer, µl Total Volume, µl
96
288
5,472
5,760
48
144
2,736
2,880
Table 7. Mixing singleplex assays.
# of Wells
20x Beads, µl Singleplex #1
20x Beads, µl Singleplex #2
Assay Buffer, µl Total Volume, µl
96
288288 5,184 5,760
48
144144 2,592 2,880
7. Run Assay
The following instructions apply to premixed multiplex, singleplex, x-Plex™,
and Express assay formats.
NOTE! Bring all buffers, diluents, diluted standards, diluted coupled
beads, and samples to room temperature prior to use. To ensure optimal
performance, pipet carefully (avoiding bubbles) with a calibrated pipet, and
use new pipet tips.
Assay Key — The terms in Table 8 are repeated throughout the assay
procedure. Refer to the detailed instructions in the table when wash and
cover and incubate are mentioned.
17
Table 8. Detailed wash and incubation instructions.
Wash
After each assay step, select the appropriate Bio-Plex Pro™ wash station program, or perform the
appropriate manual wash step as summarized below.
Bio-Plex Pro or Pro II Wash Station
Bio-Plex Pro II
Wash Station Handheld Magnet or
Vacuum Manifold
Assay Step
Magnetic Program
Vacuum Program
Manual Wash Steps
MAG x2
VAC x2
2 x 100 μl
Sample incubation
Detection Ab incubation
MAG x3
VAC x3
SA-PE incubation
3 x 100 μl
Add beads to plate
Using the Bio-Plex Pro Series Wash Stations
For detailed instructions, please refer to the quick guide (bulletin 5826) or user manual (item
#10013125). Both documents can be found at www.bio-rad.com/bio-plex.
Using the Bio-Plex® Handheld Magnetic Washer
For detailed instructions, please refer to the user guide (item #10023087), which can be
found at www.bio-rad.com/bio-plex.
Using a Vacuum Manifold
Place the filter assay plate on a calibrated vacuum apparatus and remove the buffer by
vacuum filtration. Add 100 μl wash buffer to each well and remove the liquid as before.
Ensure that all wells are exposed to the vacuum. As a general guideline, 100 μl should take
approximately 3–4 sec to completely clear the well. Repeat as specified. Thoroughly blot
the bottom of the filter plate with a clean paper towel between each vacuum step to prevent
cross contamination and plate leakage. Place the assay plate on the plastic plate holder.
COVER AND Incubate
Gently cover the plate with a new sheet of sealing tape. To prevent cross-well contamination
or leaking from the bottom of filter plates, do not press down on the tape. Place the plate on a
microplate shaker and cover with aluminum foil to prevent photobleaching. Vigorous shaking
is important. Shake at room temperature at 850 ±50 rpm for the specified incubation time.
Slowly ramp up to the maximum speed to avoid splashing of samples.
Add Coupled Beads, Samples, Standards, Blank,
and Controls
1. Cover unused wells of the assay plate with sealing tape.
2. Prewet the filter plate. Skip this step if using a flat-bottom plate.
a) Vacuum manifold: Prewet the wells with 100 µl assay buffer and
remove the liquid by vacuum filtration. Dry the bottom of the filter
plate thoroughly by blotting on a clean paper towel.
18
3. Vortex the diluted (1x) beads for 30 sec at medium speed. Pour into
a reagent reservoir and transfer 50 µl to each well of the assay plate.
TIP: A multichannel pipet is highly recommended for ease of use
and efficiency.
4.Wash the wells twice with 100 µl Bio-Plex wash buffer according to
your wash method of choice.
5.Vortex the diluted samples, standards, blank, and controls for 5
sec. Transfer 50 µl of each to the appropriate well of the assay plate,
changing the pipet tip after every volume transfer.
6. Cover and incubate in the dark for 1 hour at room temperature (RT)
with shaking at 850 ±50 rpm.
Prepare and Add Detection Antibodies
1. While the samples are incubating use Tables 9–10 or the Calculation
Worksheet on page 35 to calculate the volume of detection
antibodies and Bio-Plex detection antibody diluent needed. These
calculations include ~20% excess to compensate for transfer loss.
Detection antibodies should be prepared 10 min before use.
2. Add the required volume of Bio-Plex detection antibody diluent to a
15 ml polypropylene tube.
3. Vortex the 20x stock of detection antibodies for 15–20 sec at
medium speed, then perform a 30 sec spin to collect the entire
volume at the bottom of the tube.
4. Dilute detection antibodies to 1x by pipetting the required volume into
the 15 ml tube. Vortex.
Each well of the assay requires 1.25 μl of the 20x stock adjusted to a
final volume of 25 μl in detection antibody diluent.
19
Tables 9–10 summarize the volumes required to prepare 1x detection
antibodies from a single 20x stock. Also shown are volumes to prepare 1x
antibodies when mixing two 20x stocks from singleplex assays.
Preparing 1x detection antibodies from 20x stock
Table 9. Premixed panel or one singleplex assay.
# of Wells
20x Detection
Antibodies, µl Detection Antibody
Diluent, µl
96
145
2,755
2,900
48
73
1,377
1,450
Total Volume, µl
Table 10. Mixing singleplex assays.
# of Wells
20x Detection
Antibodies, µl
Singleplex #1
20x Detection
Antibodies, µl
Singleplex #2
Detection
Antibody
Diluent, µl
Total Volume, µl
96
145 1452,610 2,900
48
73
731,3041,450
5.After incubating the beads, samples, standards, blank, and controls,
slowly remove and discard the sealing tape.
6. W
ash the wells three times with 100 µl wash buffer according to the
wash method of choice.
7. V
ortex the diluted (1x) detection antibodies gently for 5 sec. Pour into
a reagent reservoir and transfer 25 µl to each well of the assay plate
using a multichannel pipet.
8.Cover and incubate in the dark for 30 min at RT with shaking at
850 ±50 rpm.
20
Prepare and Add Streptavidin-PE (SA-PE)
1.While detection antibodies are incubating, use Table 11 or the Calculation
Worksheet on page 35 to calculate the volume of SA-PE and assay
buffer needed. These calculations include ~25% excess to account for
transfer loss. SA-PE should be prepared 10 min before use.
2. Add the required volume of assay buffer to a 15 ml polypropylene tube.
3.Vortex the 100x stock of SA-PE for 5 sec at medium speed. Perform
a 30 sec spin to collect the entire volume at the bottom of the vial.
4.Dilute SA-PE to 1x by pipetting the required volume into the 15 ml
tube. Vortex and protect from light until ready to use.
Each well of the assay requires 0.5 μl of the 100x stock adjusted to a
final volume of 50 μl in assay buffer.
Table 11. Preparing 1x SA-PE from 100x stock.
# of Wells
100x SA-PE, µl
Assay Buffer, µl
Total Volume, µl
96
60
5,940
6,000
48
30
2,970
3,000
5.After detection antibody incubation, slowly remove and discard
the sealing tape.
6.Wash the wells three times with 100 µl of wash buffer according
to the wash method of choice.
7. Vortex the diluted (1x) SA-PE at medium speed for 5 sec. Pour
into a reagent reservoir and transfer 50 µl to each well using a
multichannel pipet.
8.Cover and incubate in the dark for 10 min at RT with shaking at
850 ±50 rpm
21
9. After the streptavidin-PE incubation step, slowly remove and discard
the sealing tape.
10. Wash the wells three times with 100 µl of wash buffer according
to the wash method of choice.
11.To resuspend beads for plate reading, add 125 µl assay buffer to
each well. Cover the plate with a new sheet of sealing tape. Shake
at RT at 850 ±50 rpm for 30 sec and slowly remove the sealing
tape. Ensure that the plate cover has been removed before placing
the plate on the reader.
8. Read Plate
Bio-Plex Manager™ software is recommended for all Bio-Plex Pro™ assay
data acquisition and analysis. Instructions for Luminex xPONENT software
are also included. For instructions using other xMAP system software
packages, contact Bio-Rad Technical Support or your regional Bio-Rad
field applications specialist.
Prepare Protocol in Bio-Plex Manager Software v 6.0
and Higher
The protocol should be prepared in advance so that the plate is read as
soon as the experiment is complete.
A protocol file specifies the analytes in the assay, the plate wells to be
read, sample information, the values of standards and controls, and
instrument settings.
Bio-Plex Manager software versions 6.0 and higher contain protocols for
most Bio-Plex® assays. Choose from available protocols or create a new
protocol. To create a new protocol, select File, then New from the main
menu. Locate and follow the steps under Protocol Settings.
22
1. Describe Protocol and enter information about the assay (optional).
2.Select Analytes and create a new panel. Visually confirm the selected
analytes and proceed to step 3.
a.
Click the Add Panel button
in the Select Analytes toolbar. Enter a new panel name. Select Bio-Plex Pro Assay Magnetic
from the assay pull-down menu. If using Bio-Plex Manager version 5.0 or lower, select MagPlex from the assay pull-down menu.
b.
Click the Add button. Enter the bead region number and name
for the first analyte. Click Add Continue to repeat for each analyte
in the assay. Refer to the bead regions in parentheses ( ) listed on
the peel-off label provided with the standards.
For reference, bead regions for human Th17 cytokine assays are
shown in Table 12.
c. Click the Add button when the last analyte has been added and
click OK to save the new panel.
d. Highlight analytes from the Available list (left) and move to the
Selected list (right) using the Add button. To move all analytes at
once, simply click the Add All button.
Table 12. Bead regions for human Th17 cytokine assays.
Analyte
Bead Region
Analyte
Bead Region
IL-1b 39
IL-23 43
lL-4 52
IL-25 55
lL-6 19
IL-31 62
IL-10 56
IL-33 46
IL-17A 76
IFN-g 21
IL-17F 44
sCD40L 29
IL-21 47
TNF-a 36
IL-22 18
IL-17A/F*
66
* Available only as a singleplex and in Express assay format.
23
e.
If some of the analytes need to be removed from the Selected
list, highlight them and select Remove. If desired, it is possible to
rename the panel by clicking on Rename Panel and entering a
new panel name.
3.Format the plate in Bio-Plex Manager™ according to the plate layout
created in Section 1 (Plan Plate Layout). To modify the plate layout,
follow the steps below (see Figure 4).
a. Select the Plate Formatting tab.
b. Select the standards icon S and drag the cursor over all the wells that contain standards. Repeat this process for
Blanks B , Controls C , and Samples X . Note that
Bio-Plex Manager™ automatically subtracts the blank FI value
from all other assay wells.
Fig. 4. Plate formatting
24
4. Enter Standards Info into Bio-Plex Manager.
a. Enter the highest concentration of each analyte in the top row
(labeled S1) of the table. S1 concentration information is included
with each vial of standards.
b. Enter a dilution factor of 4 and click Calculate. The concentrations
for each standard point will be populated for all analytes in the table.
c. Optional: enter the lot number of the vial of standards into the
Standard Lot box and click Save.
5.Enter Controls Info — for user specified controls, select an analyte
from the pull-down menu, then enter a description and concentration.
Repeat for each additional analyte in the assay.
For the quality controls supplied in premixed Th17 cytokine kits,
format the appropriate wells as controls, enter descriptions, but leave
the concentrations blank. Alternatively, the quality controls can be
formatted as samples with clear descriptions such as “quality control
high” and “quality control low”. In any case, the expected control
ranges provided on the product data sheet are not entered into
Bio-Plex Manager software v6.1 and earlier.
6. Enter Sample Info — enter sample information and the appropriate
dilution factor.
7. Run Protocol — confirm that the assay settings are correct.
a.Human Th17 cytokine assays are run at the default setting in
Bio-Plex Manager — low RP1 target (low PMT).
b.Confirm that data acquisition is set to 50 beads per region.
In Advanced Settings, confirm that the bead map is set to 100
region, the sample size is set to 50 µl, and the DD gates are set
to 5,000 (Low) and 25,000 (High). In Bio-Plex Manager software
versions 4.0, 4.1, 4.1.1, and 5.0, check Override Gates and set
the DD gate values as indicated.
Select Start, name and save the .rbx file, and begin data
acquisition. The Run Protocol pop-up screen will appear.
Click Eject/Retract to eject the plate carrier.
25
Acquire Data
1.
Shake the assay plate at 850 ±50 rpm for 30 sec, and visually
inspect the plate to ensure that the assay wells are filled with buffer.
Slowly remove the sealing tape and any plate cover before placing
the plate on the plate carrier.
2.Run Protocol – on the pop-up screen, select Load Plate and click OK
to start acquiring data.
3. Use the Wash Between Plates
command after every plate run
to reduce the possibility of clogging the instrument.
4.
If acquiring data from more than one plate, empty the waste bottle
and refill the sheath bottle after each plate (if HTF are not present).
Select Wash Between Plates and follow the instructions. Then repeat
the Prepare Protocol and Acquire Data instructions.
5. When data acquisition is complete, select Shut Down
follow the instructions.
and
Reacquire Data
It is possible to acquire data from a well or plate a second time using the
Rerun/Recovery mode located below Start in the Run Protocol step.
Any previous data will be overwritten.
1. Check the wells from which data will be reacquired.
2.Aspirate the buffer with the wash method of choice, but do not
perform wash step.
3. Add 100 µl assay buffer to each well. Cover the plate with a new
sheet of sealing tape.
4. Repeat the Acquire Data steps to reacquire data. The data acquired
should be similar to those acquired initially; however, the acquisition
time will be extended because the wells have fewer beads.
26
Data Analysis
Quality Controls
If the quality controls were run in the assay plate, open the results (.rbx) file,
click on Report Table, and locate the control wells. Visually compare the
observed concentrations of the high and low controls in the Report Table
against the lot-specific control ranges shown in the product data sheet.
Expected control ranges are provided for reference and should be used
as general guidelines. Actual results may vary for some operators. If
the controls do not fall within the expected ranges, please refer to the
troubleshooting section for possible causes and solutions.
Removing Outliers
Outliers are identified as standard data points that do not meet accuracy
or precision requirements and should be considered invalid when
performing curve fitting. As such, they should be removed to generate a
more realistic and accurate standard curve. This may result in an extended
assay working range and allow quantitation of samples that might
otherwise be considered out of range (OOR).
In Bio-Plex Manager software version 6.0 and higher, outliers can be
automatically removed by selecting the Optimize button in the Standard
Curve window. In Bio-Plex Manager software 6.0 and earlier versions,
outliers also can be manually selected in the Report Table. Visit online Help
to learn more about the standard curve optimizer feature and how outliers
are determined.
Previous Versions of Bio-Plex Manager Software
For instructions on using previous versions of Bio-Plex manager software,
please contact Bio-Rad Technical Service.
27
Luminex xPONENT Software
Luminex xPONENT software may be used to analyze Bio-Plex assays.
Although guidelines are provided here, consult the xPONENT software
manual for more details. Perform a system initialization with Luminex’s
calibration and performance verification kit, as directed by Luminex.
Select Batches to set up the protocol and follow the information
under Settings.
1. Select MagPlex as the bead type for magnetic beads. This
automatically sets the DD gates.
2. Volume = 50 µl
3. Bio-Plex Pro Human Th17 cytokine assays are run at low PMT
(Standard PMT).
4. Plate name: 96-well plate.
5. Analysis type: Quantitative, 5PL Curve Fit.
6. Number of standards: 8.
Select Analytes to set up the panel.
1. Enter “pg/ml” in the Units field.
2. Enter 50 in the Count field.
3. Select the bead region and enter the analyte name.
4. Click Apply all for Units and Count.
Select Stds and Ctrls.
1. Enter standard concentrations, lot number, dilution factor, and other
information, as applicable.
After the assay is complete, select Results, then select Saved Batches.
28
Troubleshooting Guide
This troubleshooting guide addresses problems that may be encountered
with Bio-Plex Pro™ assays. If you experience any of the problems listed
below, review the possible causes and solutions provided. Poor assay
performance may also be due to the Bio-Plex® suspension array reader.
To eliminate this possibility, use the validation kit to validate all the key
functions of the array reader and to assist in determining whether or not
the array reader is functioning properly.
Possible Causes
High Inter-Assay CV Standards and controls were not
reconstituted consistently between
assays
Reconstituted standards, controls,
and diluted samples were not
stored properly
Bottom of filter plate not dry
Possible Solutions
Incubate the reconstituted
standards for 30 min on ice. Always
be consistent with the incubation
time and temperature.
Reconstituted standards and diluted
samples should be prepared on ice
as instructed. Prior to plating, the
reconstituted standards and diluted
samples should be equilibrated to
room temperature.
Dry the bottom of the filter plate with
absorbent paper towel (preferably
lint-free) to prevent cross-well
contamination.
29
Possible Causes
Possible Solutions
High Intra-Assay CV
Improper pipetting technique
Reagents and assay components
not equilibrated to room
temperature prior to pipetting
Contamination with wash buffer
during wash steps
Pipet carefully when adding
standards, controls, samples,
detection antibodies, and
streptavidin-PE, especially when
using a multichannel pipet. Use a
calibrated pipet. Change pipet tip
after every volume transfer.
All reagents and assay components
should be equilibrated to room
temperature prior to pipetting.
During the wash steps, be careful
not to splash wash buffer from one
well to another. Be sure that the
wells are filtered completely and that
no residual volume remains. Ensure
that the microplate shaker setting is
not too high. Reduce the microplate
shaker speed to minimize splashing
Slow pipetting of samples and
reagents across the plate
Sample pipetting across the entire
plate should take less than 4 min.
Reagent pipetting across the entire
plate should take less than 1 min.
30
Possible Causes
Low Bead Count
Miscalculation of bead dilution
Possible Solutions
Beads clumped in multiplex
bead stock tube
Vortex for 30 sec at medium speed
before aliquoting beads.
Vacuum on for too long when
aspirating buffer from wells
Assay plate not shaken enough
during incubation steps and prior
to reading
Reader is clogged
Do not apply vacuum to the filter
plate for longer than 10 sec after the
buffer is completely drained from
each well.
Incorrect needle height of the
reader
Adjust the needle height to coincide
with the plate type provided in the kit.
Check your calculations and be
careful to add the correct volumes
Shake the plate at 850 ±50 rpm
during incubation steps and for
30 sec immediately before reading
the plate.
Refer to the troubleshooting guide
in the Bio-Plex system hardware
instruction manual (bulletin
#10005042).
Low Signal or Poor Sensitivity
Standards reconstituted incorrectly Follow the cytokine standard
instructions carefully.
Detection antibody or streptavidin-PE diluted incorrectly
Check your calculations and be
careful to add the correct volumes.
31
Possible Causes
Possible Solutions
Incorrect buffer was used
(for example, assay buffer used
to dilute standards)
Use sample matrix standard diluent
to dilute standards.
High Background Signal
Accidentally spiked blank wells
Detection antibodies or
streptavidin-PE incubated too long
Poor Recovery
Expired Bio-Plex reagents
were used
Incorrect amounts of components
were added
Be careful when spiking standards.
Do not add any antigens to the
blank wells.
Follow the procedure incubation
time precisely.
Check that reagents have not expired.
Use new or nonexpired components.
Check your calculations and be
careful to add the correct volumes.
Microplate shaker set to an
incorrect speed
Check the microplate shaker speed
and use the recommended setting.
Setting the speed too high may
cause splashing and contamination.
Use the recommended plate shaker.
High end saturation of the
standard curve
Make sure that correct shaker speed
and incubation times are used.
Remove S1 for data analysis if needed.
Quality controls do not fall within
expected ranges.
Make sure that the controls are
reconstituted at the same time as
standards, and in the same diuent
(standard diluent HB). Incubate for
precisely 30 min.
32
Possible Causes
Possible Solutions
Poor Recovery
Improper pipetting
technique
Impact of Sample
Matrix
Negative MFI values in
samples or standards
Pipet carefully when adding standards, samples,
detection antibodies, and streptavidin-PE, especially
when using a multichannel pipet. Use a calibrated
pipet.Change pipet tip after every volume transfer.
If samples contain little or no analyte, negative
values observed may be due to statistical variation.
If assay drift is suspected, retest the samples
by positioning them next to the standards. If
contamination of standards is suspected, check
the standard replicate value and be careful when
adding samples to the wells. Matrix effects could
also produce negative sample values.
Bio-Plex Manager™ software automatically
subtracts the Blank (B) FI value from all other
assay wells. While this has no impact on observed
concentrations of samples within the assay working
range, it may result in a negative FI value if the
Blank’s FI value is greater than either the standard
or sample value. If this is undesirable, then reformat
the blank wells as Sample (X) or Control (C), in the
protocol or results file.
Poor precision in
serum and
plasma sample
measurements
Check if any interfering components such as
heparin-based anticoagulant, additives, or
gel from separators were introduced into the
samples. Avoid using hemolyzed and heavily
lipemic samples. Remove visible particulate in
samples by centrifugation. Avoid multiple freezethaw cycles of samples.
33
Plate Layout Template
34
Calculation Worksheet
If using either a premixed panel or one singleplex assay, follow these directions.
Plan the plate layout and enter the number of wells to be used in the assay:_______
1
1. Determine the volume of 1x coupled beads needed.
a. Each well requires 50 µl of coupled beads (1x): _______ x 50 µl = _______ µl
12
b. Include a 20% excess to ensure enough volume: _______ µl x 0.20 = _______ µl
23
c. Total volume of 1x coupled beads: _______ µl + _______ µl = _______ µl
234
d. Volume of 20x coupled beads stock: _______ µl /20 = _______ µl
45
e. Volume of assay buffer required: _______ µl – _______ µl = _______
45 6
2. Determine the volume of 1x detection antibody needed.
a. Each well requires 25 µl detection antibodies (1x): ______ x 25 µl = _______ µl
17
b. Include a 20% excess to ensure enough volume: _______ µl x 0.2 = _______ µl
78
c. Total volume of 1x detection antibodies: _______ µl + _______ µl = _______ µl
78 9
d. Volume of 20x detection antibodies stock: _______ µl /20 = _______ µl
910
e. Volume of detection antibody diluent required: _____ µl – _____ µl = _____ µl
9 1011
3. Determine the volume of 1x streptavidin-PE needed.
a. Each well requires 50 µl streptavidin-PE (1x): _______ x 50 µl = _______ µl
110
b. Include 25% excess to ensure enough volume: _______ µl x 0.25 = _______ µl
1011
c. Total volume of 100x streptavidin-PE: ______ µl + ______ µl = ______ µl
1011 12
d. Volume of 100x streptavidin-PE required: _______ µl / 100 = _______ µl
1213
e. Volume of assay buffer required: _______ µl – _______ µl = _______ µl
1213 14
35
If mixing singleplex assays, follow these directions.
Enter the number of wells to be used in the assay:_______
1
1. Determine the volume of 1x coupled beads needed.
a. Each well requires 50 µl coupled beads (1x): _______ x 50 µl = _______ µl
12
b. Include 20% excess to ensure enough volume: _______ µl x 0.20 = _______ µl
23
c. Total volume of 1x coupled beads: _______ µl + _______ µl = _______ µl
23 4
d. Enter the number of singleplex sets (or analytes) that will be multiplexed: _______
5
e. Volume of 20x coupled beads required from each coupled beads tube:
_______ µl / 20 = _______ µl
46
f. Total volume of bead stock required: _______ x _______ µl = _______ µl
56 7
g. Volume of assay buffer required: _______ µl – _______ µl = _______ µl
478
2. Determine the volume of 1x detection antibody needed.
a. Each well requires 25 µl detection antibodies (1x): _______ x 25 µl = _______ µl
19
b. Include a 20% excess to ensure enough volume: _______ µl x 0.2 = _______ µl
910
c. Total volume of 1x detection antibodies: _______ µl + _______ µl = _______ µl
9
1011
d. Enter the number of singleplex sets (or analytes) that will be multiplexed: _______
5
e. Volume of 20x detection antibodies required from each detection antibody tube:
_______ µl / 20 = _______ µl
1112
f. Total volume of detection antibody stock: _____ µl x _____ = _____ µl
12 513
g. Volume of detection antibody diluent required: ____ µl – ____ µl = ____µl
111314
3. Determine the volume of 1x streptavidin-PE needed.
a. Each well requires 50 µl streptavidin-PE (1x): _______ x 50 µl = _______ µl
115
b. Include 25% excess to ensure enough volume: _______ µl x 0.25 = _______ µl
1516
c. Total volume of 100x streptavidin-PE: ______ µl + ______ µl = _______ µl
1516 17
d. Volume of 100x streptavidin-PE required: _______ µl / 100 = _______ µl
1718
e. Volume of assay buffer required: _______ µl _______ µl = _______ µl
1718 19
36
Safety Considerations
Eye protection and gloves are recommended when using these products.
Consult the MSDS for additional information. The Bio-Plex Pro™ assays
contain components of animal origin. This material should be handled as
if capable of transmitting infectious agents. Use universal precautions.
These components should be handled at Biosafety Level 2 containment
as defined by U.S. government publication, Biosafety in Microbiological
and Biomedical Laboratories (Centers for Disease Control, 1999).
Legal Notices
Acrodisc and Supor are trademarks of Pall Corporation. MagPlex, xMAP,
xPONENT, and Luminex are trademarks of Luminex Corporation.
The Bio-Plex® suspension array system includes fluorescently labeled
microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc.
by the Luminex Corporation.
37
Ordering Information
Catalog #
Premixed Multiplex Assay Panel
171-AA001M Bio-Plex Pro Human Th17 Cytokine Panel 15-plex, 1 X 96-well
Human Th17 Cytokine Singleplex Sets, 1 x 96 well (requires reagent kit II and standards)
171-BA001MIL-1b
171-BA002MlL-4
171-BA003MlL-6
171-BA004MIL-10
171-BA005MIL-17A
171-BA006MIL-17F
171-BA007MIL-21
171-BA008MIL-22
171-BA009MIL-23
171-BA010MIL-25
171-BA011MIL-31
171-BA012MIL-33
171-BA013MIFN-g
171-BA014MsCD40L
171-BA015MTNF-a
171-BA016M
IL-17A/F Individual Components and Accessories
171-304055 Bio-Plex Pro Reagent Kit II, 1 x 96-well Filter Plate
171-304055M Bio-Plex Pro Reagent Kit II, 1 x 96-well Flat-Bottom Plate
171-DA0001
Bio-Plex Pro Human Th17 Cytokine 16-plex Standards, pkg of 1 vial
171-DA0501
Bio-Plex Pro Human Th17 Cytokine 16-plex Standards, pkg of 50 vials
171-304502 Filter Plate, 1 x 96-well with clear plastic lid and tray
Bio-Plex® x-Plex™ Assays (We Mix)
Premium, custom assay service using the Bio-Plex Assay Builder,
www.bio-rad.com/bio-plex/assaybuilder, to select analytes and plate type
of interest. Assays are supplied as premixed coupled beads and detection
antibodies in the all-in-one kit format. Quality controls are included.
Bio-Plex Express Assays (You Mix)
Fast and economical custom assay service using the Bio-Plex Assay Builder,
www.bio-rad.com/bio-plex/assaybuilder, to select analytes and plate
type of interest. Assays are supplied as individual sets of coupled beads and
detection antibodies in the all-in-one kit format. Quality controls not included.
Other Components and Accessories
Wash buffer, Bio-Plex Pro flat-bottom plates, and streptavidin-PE are also
available individually. For more information, go to www.bio-rad.com/bio-plex.
38
39
Bio-Rad
Laboratories, Inc.
Life Science
Group
10023381 Rev A
Web site www.bio-rad.com USA 800 424 6723
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Brazil 55 11 5044 5699 Canada 905 364 3435 China 86 21 6169 8500
Czech Republic 420 241 430 532 Denmark 44 52 10 00
Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0
Greece 30 210 9532 220 Hong Kong 852 2789 3300
Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050
Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460
Mexico 52 555 488 7670 The Netherlands 0318 540666
New Zealand 64 9 415 2280 Norway 23 38 41 30
Poland 48 22 331 99 99 Portugal 351 21 472 7700
Russia 7 495 721 14 04 Singapore 65 6415 3188
South Africa 27 861 246 723 Spain 34 91 590 5200
Sweden 08 555 12700 Switzerland 061 717 95 55
Taiwan 886 2 2578 7189 Thailand 800 88 22 88
United Kingdom 020 8328 2000
Sig 1211
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