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Bio-Plex Pro
™
Human
TIMP 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.
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Table of Contents
Introduction 1
Principle 2
Kit Contents and Storage 4
Recommended Materials 5
Assay Workflow 6
lmportant Considerations 7
Detailed Instructions 7
1.1. Plan Plate Layout 8
2.2. Prepare Instrument 9
3.3. Prepare Wash Method 10
4.4. Prepare Wash Buffer 11
5.5. Prepare Standards and Controls 11
6. Prepare Samples 14
7.7. Prepare Coupled Beads 16
8.8. Run Assay 17
9.9. Read Plate 21
Troubleshooting Guide 28
Plate Layout Template 33
Calculation Worksheet 34
Safety Considerations 35
Legal Notices 35
Ordering Information 36
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Introduction
Bio-Plex Pro™ Human TIMP Assays
The matrix metalloproteinases (MMPs) are a family of zinc proteases
with essential roles in breaking down components of the extracellular
matrix (ECM). The MMPs are inhibited by specific endogenous tissue
inhibitors of metalloproteinases (TIMPs), which are a family of four protease
inhibitors: TIMP-1, TIMP-2, TIMP-3, and TIMP-4. The balance between
MMP and TIMP levels is crucial for the timely degradation of ECM.
MMPs and TIMPs play important roles in tissue remodeling associated
with various physiological and pathological processes such as
inflammation, apoptosis, morphogenesis, angiogenesis, tumor invasion,
and metastasis. The new MMP and TIMP panels will expand the
Bio-Plex Pro assay menu in research areas including cancer, autoimmune
disease, wound healing, and cardiovascular disease.
Advantages of Magnetic Bead-Based Assays
Products in the Bio-Plex Pro family of assays are on magnetic polystyrene
beads. These beads provide a choice in the method of assay preparation —
standard or magnet-based. The standard workflow for Bio-Plex assay
preparation requires multiple wash steps in which the 96-well filter plate
is placed on a vacuum manifold to draw the liquid through the bottom of
the filter plate. In contrast, magnet-based assay preparation permits
liquid removal from the top of the well and thus does not require a filter
plate or vacuum manifold. As a result, either an automated or manual
washer can be used. Bio-Rad offers both solutions with the automated
Bio-Plex Pro wash station and the manual Bio-Plex handheld magnetic
washer. Magnetic separation offers greater convenience and reproducibility
compared to vacuum filtration.
For a current listing of Bio-Plex assays, panels, and reagents, please visit
www.bio-rad.com/bio-plex.
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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. In
the Bio-Plex
®
MAGPIX™ system, the sample is injected into a chamber
where the beads are imaged using LED and CCD technology
n
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 (streptavidin-PE or SA-PE)
conjugate. Phycoerythrin serves as a fluorescent indicator, or reporter.
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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. The concentration of analyte
bound to each bead is proportional to the MFI of the reporter signal.
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Kit Contents and Storage
Reagents Supplied
Bio-Plex Pro
™
TIMP 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 kits.
Component
Diluent HD
Assay buffer
Wash buffer (10x)
Detection antibody diluent HB
Streptavidin-PE (100x)
Assay plate (96-well flat bottom plate)
Sealing tape
Assay quick guide
Product data sheet
Coupled magnetic beads (20x)
Detection antibodies (20x)
Standard
Control
* Volumes shown are approximate.
1 bottle, 180 ml
1 bottle, 50 ml
1 bottle, 60 ml
1 bottle, 5 ml
Quantity
1 tube
1 plate
1 pack of 4
1 booklet
1
1 tube
1 tube
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.
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Table 2. Recommended materials.
Item
Bio-Plex® 200 system or Luminex system with HTF
Bio-Plex validation kit
Ordering Information
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 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
®
Titertube
For preparing replicate standards, samples, and controls
micro test tubes
Bio-Rad catalog #223-9390
prior to loading the plate
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-Plex Manager
MP Software Upgrade
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
Bio-Rad catalog #171-051555
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).
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Assay Workflow
Prewet wells
(for lter plate only)
Add 50 µl 1x beads to wells
Wash 2 x 100 μl
Add 50 μl diluted 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
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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 9).
Assay Procedures
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 includes a printed Bio-Plex Pro Assay Quick Guide (bulletin
#10041638), 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
Bead regions for all analytes are listed in the Read Plate section.
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.
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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 33 or the Plate
Formatting tab in Bio-Plex Manager
is shown 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 diluent HD or a diluent similar to your final sample type or
matrix. Note that Bio-Plex Manager automatically subtracts the blank
(B) MFI value from all other assay wells.
3. Controls, either user-specified or the controls supplied, 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, calculate the required volumes
of beads, detection antibody, and streptavidin-PE needed. Use Tables 6,
8, and 9, respectively, or the Calculation Worksheet on page 34.
Legend
S Standard
™
software. A suggested plate layout
B Blank
X Samples
C Controls
Fig. 2. Suggested plate layout. For detailed instructions on plate
formatting in Bio-Plex Manager software, see section Read Plate.
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2. Prepare Instrument
These directions are specific for the Bio-Plex® 100/200 reader. To prepare
either a Bio-Plex 3D or Bio-Plex
user manuals.
Note: While the instrument is warming up, bring the 10x wash buffer,
assay buffer, and diluent HD to room temperature. Keep other items on ice
until needed. Also, begin to thaw frozen samples.
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.
®
MAGPIX™ reader, consult their respective
™
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
for 4 hr without acquiring data, the lasers will automatically turn off.
To reset the 4-hr countdown, select Warm up
lasers/optics to reach operational temperature.
and follow the instructions. If the system is idle
and wait for the
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.
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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.
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 handheld magnetic washer
Vacuum filtration Vacuum manifold (manual) Filter plate
Setting Up the Bio-Plex Pro 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 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.
™
Flat bottom plate
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).
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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.
4. Prepare Wash Buffer
1. Bring the 10x stock solution to room temperature.
2. If crystals exist, ensure that they are completely dissolved. Mix the
10x stock solution by inversion before preparing the 1x wash buffer.
3. To prepare 1x wash buffer, dilute 1 part 10x stock solution with
9 parts deionized water.
5. Prepare Standards and Controls
General Instructions
n
It is essential to prepare standards and controls 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 provided lists the most concentrated point on the
standard curve (S1). Enter the values and units into Bio-Plex Manager
software as instructed in section 9
™
Using the Controls (optional)
One vial of controls is included. Its use is intended for monitoring the
day-to-day quality of assay results.
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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
®
diluent HD. 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 samples.
Sample Type Diluent for Standard and Controls* Add BSA
Serum and plasma Diluent HD None
Culture media, with serum Culture media None
Culture media, serum-free Culture media To 0.5% final
* If using diluents other than the diluent HD provided, then users must establish their own
control ranges.
Reconstitute Standards and Controls
This procedure prepares enough standard to run each dilution in duplicate.
Note: The appearance of the lyophilized standards or controls may vary
from a white pellet to clear crystals. Regardless of appearance, the vials
have passed QC specifications and perform accordingly.
1. Gently tap the vial of lyophilized standards on a solid surface to
ensure that all material is at the bottom of the vial.
2. Reconstitute the vial of standards with 781 µl of the appropriate diluent.
Optional: at the same time, reconstitute the vial of controls with 250 µl
of the appropriate diluent as summarized in Table 4. Controls do not
require further dilution. If using diluents other than the diluent HD
provided, then users must establish their own control ranges.
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.
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Prepare the Standard Dilution Series
The following procedure produces an eight-point standard curve with
a threefold dilution between each point. Pipet carefully using calibrated
pipets and use a new pipet tip for every volume transfer.
1. Label eight 1.5 ml microcentrifuge tubes S2 through S8 and Blank.
Alternatively, using Titertube
convenient if a multichannel pipet will be used to load the plate.
2. Add 150 µl of the appropriate diluent to tubes S2–S8 and Blank.
(Figure 3).
3. Vortex reconstituted standards at medium speed for 5 sec before
removing any volume. Transfer 75 µl to the S2 tube containing the
chosen standard diluent. Vortex for 5 sec.
4. Use a new pipet tip to transfer 75 µl from the S2 tube to the S3 tube.
Vortex for 5 sec.
5. Continue with 1:3 (threefold) 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 threefold dilution series with a single reconstituted standard.
75 75 75 75 75 75 75
®
micro test tubes may prove to be more
Transfer Volume, µl
Reconstituted
Standard
150 150 150 150 150 150 150 150
S1 S2 S3 S4 S5 S6 S7 S8 Blank
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Diluent, µl
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6. Prepare Samples
These assays are designed to quantitate classes and subclasses of
immunoglobulins in serum, plasma, and cell culture media. For optimal
recovery and sensitivity, it is important to properly prepare samples.
n
Once thawed, keep samples on ice. Prepare dilutions just prior to the
start of the assay and equilibrate to room temperature before use
n
Prepare sample dilutions in 1.5 or 2 ml microcentrifuge tubes. If a
multichannel pipet will be used to load the plate, aliquot the required
volumes into Titertube
n
Do not freeze diluted samples
Table 5. Summary of recommended sample diluents and dilution factors.
Sample Type Dilution Factor* Recommended Sample Dilution
Serum and plasma TIMP: 1:50 dilution Diluent HD
Fluids TIMP: 1:4 and 1:40 Diluent + 0.5% BSA w/v
* TIM-3 is not intended for serum and plasma samples.
Sample Preparation
Serum and Plasma
EDTA or citrate is preferred as an anticoagulant. Heparin-treated plasma,
while compatible with Bio-Plex Pro
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.
®
micro test tubes
™
assays, may absorb certain soluble
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3. Perform centrifugation at 1,000 x g for 15 mi n 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. Alternatively, filter the samples with a
0.8/0.2 m dual filter to prevent clogging.
5. Dilute 5 µl sample with 245 µl diluent HD for a 1:50 dilution.
Vortex for 5 sec.
6. Assay samples immediately or aliquot into single-use tubes and
store at –70°C. Avoid repeated freeze-thaw cycles.
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 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. Reconstitute and dilute the standard in the same medium or
matrix in which cells are prepared. Be sure to include all medium
components (such as FBS) as appropriate. To minimize error due
to lot-to-lot variation of culture media, use the same lot of culture
medium that was used to prepare the cells.
4. Assay immediately or store samples in single-use aliquots at –70°C.
Avoid repeated freeze-thaw cycles.
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7. Prepare Coupled Beads
1. Use Table 6 or the Calculation Worksheet on page 34 to calculate the
volume of coupled beads and assay buffer needed.
2. Add the required volume of Bio-Plex
polypropylene tube.
3. Vortex the 20x stock of coupled beads at medium 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 l of the 20x stock adjusted to a
final volume of 50 l in assay buffer.
5. Protect the beads from light with aluminum foil. Equilibrate to room
temperature prior to use.
Note: To minimize volume loss, use a 200–300 l capacity pipet to
remove beads from the 20x stock tube. If necessary, perform the volume
transfer in two steps. Do not use a 1,000 l capacity pipet and/or wide
bore pipet tip.
Table 6. Preparing 1x coupled beads from 20x stock (includes 20% excess volume).
# of Wells 20x Beads, µl Assay Buffer, µl Total Volume, µl
96 288 5,472 5,760
48 144 2,736 2,880
®
assay buffer to a 15 ml
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8. Run Assay
Considerations
n
Bring all buffers, diluents, diluted standards, diluted coupled beads, and
samples to room temperature before use
n
Use calibrated pipets and pipet carefully, avoiding bubbles. Use a new
pipet tip for every volume transfer
n
Pay close attention to vortexing, shaking, and incubation instructions.
Deviation from the protocol may result in low assay signal and
assay variability
n
Assay incubations are carried out in the dark at 850 ± 50 rpm. Cover
the plate with sealing tape and protect from light with aluminum foil
Table 7. Summary of wash steps and incubations. After each assay step, select the
appropriate Bio-Plex Pro
as summarized below.
Bio-Plex Pro Handheld Magnet or
Wash Station Wash Station Vacuum Manifold
Assay Step Magnetic Program Vacuum Program Manual Wash Steps
Add beads to plate MAG x2 VAC x2 2 x 100 l
Sample incubation
Detection Ab incubation MAG x3 VAC x3 3 x 100 l
SA-PE incubation
™
wash station program or perform the appropriate manual wash step
Considerations When Using a Vacuum Manifold
n
After each incubation, place the filter plate on a calibrated vacuum
apparatus and remove the liquid by vacuum filtration
n
To wash, add 100 l wash buffer to each well and remove the liquid as
before. Ensure that all wells are exposed to the vacuum
n
Thoroughly blot the bottom of the filter plate with a clean paper towel
between each vacuum step to prevent cross contamination
n
Place the assay plate on the plastic plate holder/tray as needed
n
Before each incubation, gently cover the plate with a new sheet of sealing
tape. Avoid pressing down over the wells to prevent leaking from the bottom
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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) 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.
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 plate two times with 100 µl Bio-Plex
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 with a new sheet of sealing tape and incubate in the dark for
1 hr at room temperature with shaking at 850 ± 50 rpm.
®
wash buffer
Prepare and Add Detection Antibodies
1. While the samples are incubating use Table 8 or the Calculation
Worksheet on page 34 to calculate the volume of detection
antibodies and Bio-Plex detection antibody diluent needed. 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 sec at medium
speed, then perform a 30 sec spin to collect the entire volume at the
bottom of the tube.
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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 l of the 20x stock adjusted to a
final volume of 25 l in detection antibody diluent.
Table 8. Preparing 1x detection antibodies from 20x stock (includes 25% excess volume).
20x Detection Detection Antibody
# of Wells Antibodies, µl Diluent, µl Total Volume, µl
96 150 2,850 3,000
48 75 1,425 1,500
5. After incubating the beads, samples, standards, blank, and controls,
slowly remove and discard the sealing tape.
6. Wash the plate three times with 100 µl wash buffer according to the
wash method of choice.
7. Vortex 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 with a new sheet of sealing tape and incubate in the dark for
30 min at room temperature with shaking at 850 ± 50 rpm.
Prepare and Add Streptavidin-PE (SA-PE)
1. While detection antibodies are incubating, use Table 9 or the Calculation
Worksheet on page 34 to calculate the volume of SA-PE and assay
buffer needed. 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.
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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 l of the 100x stock adjusted to a
final volume of 50 l in assay buffer.
Table 9. Preparing 1x SA-PE from 100x stock (includes 25% excess volume).
# 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 plate 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 with a new sheet of sealing tape and incubate in the dark for
10 min at room temperature with shaking at 850 ± 50 rpm.
9. After the streptavidin-PE incubation step, slowly remove and discard
the sealing tape.
10. Wash the plate 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 room temperature 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.
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9. 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
Version 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
protocol. To create a new protocol, select File, then New from the main
menu. Locate and follow the steps under Protocol Settings.
1. Click Describe Protocol in the Protocol Settings bar and enter
information about the assay (optional).
2. Click Select Analytes and create a new panel. Visually confirm the
selected analytes and proceed to step 3.
a. Click the Add Panel button
®
assays. Choose from available protocols or create a new
in the Select Analytes toolbar.
Enter a new panel name. Select Bio-Plex Pro Assay Magnetic
from the assay dropdown list. If using Bio-Plex Manager version
5.0 or lower, select MagPlex from the assay dropdown list.
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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.
For reference, bead regions are shown in Table 10.
Table 10. Bead regions for Bio-Plex Pro TIMP assays.
Analyte Bead Region Analyte Bead Region
TIMP-1 21 TIMP-3 52
TIMP-2 64 TIMP-4 35
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.
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. Click Format Plate and format according to the plate template
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.
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Fig. 4. Plate formatting.
4. Click Enter Standards Info.
a. Enter the highest concentration and units of each analyte in the
top row (labeled S1) of the table. S1 concentration information is
listed in the product data sheet.
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.
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5. Click Enter Controls Info.
a. For user-specified controls, select an analyte from the dropdown
menu, then enter a description and concentration. Repeat for
each additional analyte in the assay.
b. For the vial of controls supplied, format the appropriate wells
as controls and enter descriptions but leave the concentrations
blank. Alternatively, the controls can be formatted as samples
with clear descriptions such as “control.” In any case, the
expected control ranges provided on the product data
sheet are not entered into Bio-Plex Manager software
version 6.1 and earlier.
6. Click Enter Sample Info and enter sample information and the
appropriate dilution factor.
7. Click Run Protocol and confirm that the settings follow Table 11.
Table 11. Read the plate using the appropriate instrument settings.
Instrument RP1 (PMT) DD Gates Bead Events
Bio-Plex 100, 200* Low 5,000 (low), 25,000 (high) 50
Bio-Plex 3D* Standard Select MagPlex beads 50
®
Bio-Plex
MAGPIX™ N/A, use default instrument settings
* A similar Luminex-based system may be used.
a. Confirm that data acquisition is set to 50 beads per region.
b. In Bio-Plex Manager software prior to 6.1, go to Advanced
Settings and confirm that the bead map is set to 100 region, the
sample size is set to 50 µl, and the doublet discriminator (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.
c. 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.
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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
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.
command after every plate run
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 of 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.
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Data Analysis
Controls
If the controls were run in the assay plate, open the results (.rbx) file,
click on Report Table, and locate the control wells. Compare the
observed concentrations against the lot-specific control ranges in the
product data sheet.
Note: 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.
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 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 Support.
In Depth Data Analysis
To better understand your data’s biological significance, consider using
Bio-Plex Data Pro
comparison easier. This software offers various graphing options and
automatic calculations with simple, conservative statistical calculations.
Contact your local Bio-Rad representative for more information.
26
™
software, which makes data management and
Page 29
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.
Note: The instrument settings described below apply to Luminex 100/200
and FLEXMAP or Bio-Plex 3D instruments. For the Bio-Plex MAGPIX
reader, use the default instrument settings.
1. Select MagPlex as the bead type for magnetic beads. This
automatically sets the DD gates.
2. Volume = 50 µl.
3. Refer to Table 11 to select the appropriate PMT setting for your
instrument.
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 “ng/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.
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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 determine whether the
array reader is functioning properly.
Possible Causes
High Inter-Assay CV
Standards and controls were not
reconstituted consistently between
assays
Possible Solutions
Incubate the reconstituted
standards for 30 min on ice. Always
be consistent with the incubation
time and temperature.
Reconstituted standards, controls,
and diluted samples were not
stored properly
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.
Bottom of filter plate not dry
Dry the bottom of the filter plate with
absorbent paper towel (preferably
lint-free) to prevent cross-well
contamination.
28
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Possible Causes
High Intra-Assay CV
Improper pipetting technique
Possible Solutions
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.
Reagents and assay components
not equilibrated to room
temperature prior to pipetting
Contamination with wash buffer
during wash steps
Slow pipetting of samples and
reagents across the plate
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.
Sample pipetting across the entire
plate should take less than 4 min.
Reagent pipetting across the entire
plate should take less than 1 min.
29
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Possible Causes
Low Bead Count
Miscalculation of bead dilution
Possible Solutions
Check your calculations and be
careful to add the correct volumes.
Beads clumped in multiplex
bead stock tube
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
Incorrect needle height of the
reader
Low Signal or Poor Sensitivity
Standards reconstituted incorrectly
Detection antibody or
streptavidin-PE diluted incorrectly
Vortex for 30 sec at medium speed
before aliquoting beads.
Do not apply vacuum to the filter
plate for longer than 10 sec after the
buffer is completely drained from
each well.
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).
Adjust the needle height to coincide
with the plate type provided in the kit.
Follow the instructions carefully.
Check your calculations and be
careful to add the correct volumes.
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Possible Causes
High Background Signal
Incorrect buffer was used
(for example, assay buffer used
to dilute standards)
Possible Solutions
Use diluent HD to dilute standards.
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
Microplate shaker set to an
incorrect speed
High end saturation of the
standard curve
Controls do not fall within
expected ranges.
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.
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.
Make sure that correct shaker speed
and incubation times are used. Remove
S1 from data analysis if needed.
Make sure that the vial of controls
is reconstituted at the same time as
standards and in the same diluent.
Incubate for precisely 30 min.
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Page 34
Possible Causes
Poor Recovery
Improper pipetting
technique
Impact of Sample Matrix
Negative MFI values in
samples or standards
Possible Solutions
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
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 the 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.
32
™
software automatically
Page 35
Plate Layout Template
33
Page 36
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
b. Include 20% excess to ensure enough volume: _______ µl x 0.20 = _______ µl
c. Total volume of 1x coupled beads: _______ µl + _______ µl = _______ µl
d. Volume of 20x coupled beads required: _______ µl/20 = _______ µl
e. Volume of assay buffer required: _______ µl – _______ µl = _______ µl
2 3 4
4 5 6
2. Determine the volume of 1x detection antibody needed.
a. Each well requires 25 µl detection antibodies (1x): ______ x 25 µl = _______ µl
b. Include 25% excess to ensure enough volume: _______ µl x 0.25 = _______ µl
c. Total volume of 1x detection antibodies: _______ µl + _______ µl = _______ µl
d. Volume of 20x detection antibodies required: _______ µl/20 = _______ µl
e. Volume of detection antibody diluent required: _____ µl – _____ µl = _____ µl
3. Determine the volume of 1x streptavidin-PE needed.
a. Each well requires 50 µl streptavidin-PE (1x): _______ x 50 µl = _______ µl
b. Include 25% excess to ensure enough volume: _______ µl x 0.25 = _______ µl
c. Total volume of 1x streptavidin-PE: ______ µl + ______ µl = ______ µl
d. Volume of 100x streptavidin-PE required: _______ µl/100 = _______ µl
e. Volume of assay buffer required: _______ µl – _______ µl = _______ µl
10 11 12
12 13 14
1 2
2 3
4 5
1 7
7 8
7 8 9
9 10
9 10 11
1 10
10 11
12 13
34
Page 37
Safety Considerations
Eye protection and gloves are recommended when using these products.
Consult the MSDS for additional information. Bio-Plex Pro
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 the U.S. government publication, Biosafety in Microbiological
and Biomedical Laboratories (Centers for Disease Control, 1999).
™
assays
Legal Notices
Acrodisc and Supor are trademarks of Pall Corporation. MagPlex,
xMAP, xPONENT, MAGPIX, FLEXMAP 3D, and Luminex are trademarks
of Luminex Corporation.
The Bio-Plex
microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc.
by the Luminex Corporation.
®
suspension array system includes fluorescently labeled
35
Page 38
Ordering Information
Detailed ordering information can be found at www.bio-rad.com/bio-plex.
Catalog # Premixed All-In-One Multiplex Kit
Premixed multiplex kit includes coupled magnetic beads, detection antibodies, standards, 1-level controls,
detection antibody diluent HB, diluent HD (for use with samples, standards, and controls), assay buffer, wash
buffer, streptavidin-PE, 96-well flat bottom plate, sealing tape, assay quick guide, and product data sheet
171-AM002M Bio-Plex Pro Human TIMP Panel, 4-plex, 1 x 96-well, for the detection of TIMP-1,
Accessoriess
171-025001 Bio-Plex Pro Flat Bottom Plates, 40 x 96-well plates
171-304500 Bio-Plex Wash Buffer, 1.5 L
171-304502 Filter plate, pkg of 1, 96-well plate with clear plastic lid and tray, for Bio-Plex assays
171-020100 Bio-Plex Handheld Magnetic Washer, includes magnetic washer and adjustment hex
TIMP-2, TIMP-3, TIMP-4
using the vacuum wash method, sealing tape not included
tools for use in manual wash steps for all Bio-Plex magnetic assays
36
Page 39
Bio-Rad
Laboratories, Inc.
Life Science
Group
Web site ww w.bio-rad.com USA 800 424 6723
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Brazi l 55 11 3065 7550 Canada 905 364 3 435 China 86 21 6169 8500
Czech R epubli c 420 241 430 532 Den mark 44 52 10 00
Finland 09 804 22 00 France 01 47 95 69 65 Ger many 089 31 88 4 0
Greece 30 210 9532 220 Hon g Kong 852 2789 33 00
Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050
Italy 39 02 216091 Japan 81 3 6361 7000 Korea 82 2 3473 4460
Mexico 52 5 55 488 7670 The Netherlands 0318 540666
New Zealand 64 9 415 2280 No rway 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 3 4 91 590 5200
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Sig 121310041640 Rev A
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