10028258
Bio-Plex Pro RBM
Kidney Toxicity 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.
FPO
Table of Contents
Introduction1
Principle2
Kit Contents and Storage
4
Recommended Materials
5
Assay Workflow
6
lmportant Considerations
7
Detailed Instructions 7
1. Plan Plate Layout8
2. Prepare Instrument9
3. Prepare Wash Method10
4. Prepare Reagents11
5. Prepare Samples13
6. Run the Assay14
7. Read Plate16
Troubleshooting Guide
23
Plate Layout Template
28
Safety Considerations
29
Legal Notices
29
Ordering Information
30
Introduction
Early and Accurate Detection of Kidney Toxicity
Acute kidney injury (AKI) is frequently caused by trauma to the kidney due
to drug exposure, an accident, or blood loss from a medical procedure.
In contrast, chronic kidney injury (CKI) results from long-term diseases
such as diabetes, high blood pressure, or from an inherited syndrome.
Early detection of AKI along with proper treatment regimens is critical
in the prevention of further loss of kidney function. The Bio-Plex Pro™
RBM kidney toxicity assays, developed in partnership with Myriad RBM,
comprise a highly relevant set of biomarkers, found in urine, for early
detection and characterization of kidney toxicity or injury. Myriad RBM’s
close collaboration with the Predictive Safety Testing Consortium (PSTC)
was instrumental in the selection of markers found in these panels.
Traditional markers such as serum creatinine (SCr) and blood urea
nitrogen (BUN) are detectable days or weeks after kidney damage has
occurred. The Bio-Plex Pro RBM kidney toxicity assays provide a tool to
detect biomarkers that may reveal damage within hours, allowing drugs to
be efficiently tested in both preclinical and clinical research settings.
Bio-Plex Pro RBM Kidney Toxicity Assays
The Bio-Plex Pro RBM assays are built on magnetic beads to enable
robust quantification of multiple proteins in human, canine, and rat urine
samples. For researchers working with limited sample volume, the
capacity to multiplex in a small sample volume provides an effective option
over the traditional ELISA. 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, productivity, and
reproducibility compared to vacuum filtration.
For more information please visit www.bio-rad.com/bio-plex.
1
Principle
Technology
The Bio-Plex® multiplex system is built upon the three core elements of
xMAP technology:
n
n
n
Fluorescently dyed magnetic 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 up to 500 different types of molecules in a single well of
96-well microplate on the Bio-Plex® 3D system, up to 100 different
types of molecules on the Bio-Plex® 200 system, and up to 50 different
types of molecules on the Bio-Plex® MAGPIX™ system
On the Bio-Plex 200 and Bio-Plex 3D systems, 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, the entire sample load volume is injected into a
chamber where the beads are imaged using LED and CCD technology
A high-speed digital signal processor that efficiently manages the
fluorescence data
Assay Format
The Bio-Plex Pro™ RBM kidney toxicity 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
Fig. 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 the reporter signal.
3
Kit Contents and Storage
Reagents Supplied
The Bio-Plex Pro™ RBM kidney toxicity assays are available in a
convenient kit format that includes assay, reagent, and diluent
components in a single box (Table 1).
Table 1. Contents of 1 x 96-well kits.
Component
Quantity
Volume
Volume After
Reconstitution or Dilution
Capture beads (1x)
1 tube
1.4 ml
Detection antibodies
1 vial
Lyophilized
Standards mix
1 vial
Lyophilized
150 μl
Control 1 (high)
1 vial
Lyophilized
100 μl
4.8 ml
Control 2 (low)
1 vial
Lyophilized
100 μl
Blocking buffer
1 vial
Lyophilized
1.5 ml
Standard diluent
1 vial
Lyophilized
1.0 ml
Sample dilution buffer
1 bottle
Assay buffer (10x)
1 bottle
60 ml
600 ml
Streptavidin-PE (10x)
1 tube
250 μl
2.5 ml
Assay plate (96-well flat bottom)
1 plate
Plate seals
1 pack of 3
Assay quick guide
1 sheet
Product data sheet
1 sheet
100 ml
Storage and Stability
Kit contents should be stored at 2–8°C and never frozen. Coupled
magnetic beads and streptavidin-PE should be stored in the dark. All
components are guaranteed for a minimum of six months from the date of
purchase when stored as specified.
4
Table 2. Recommended materials.
Item
Ordering Information
Bio-Plex® 200 system or Luminex system with HTF
Bio-Rad catalog #171-000205
Bio-Plex validation kit
Note: Run the validation kit monthly to ensure optimal
performance of fluidics and optics systems
Bio-Rad catalog #171-203001
Bio-Plex calibration kit
Note: Run the calibration kit daily 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 x 96-well)
For magnetic separation on the Bio-Plex Pro wash station
Bio-Rad catalog #171-025001
Titertube® micro test tubes
For preparing replicate standards, samples, and controls
prior to loading the plate
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)
Bio-Rad catalog #223-9390
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, 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
Reconstitute lyophilized reagents, dilute assay
buffer to 1x, prepare standards and samples
Add 10 µl blocking buffer to all wells
Add 30 μl standards, blank, samples,
and controls to appropriate wells
Add 10 μl 1x capture beads per well.
Incubate at 850 ± 50 rpm for 1 hr at RT
Wash 3x with 100 µl assay buffer (1x)
Add 40 μl reconstituted detection antibodies.
Incubate at 850 ± 50 rpm for 1 hr at RT
Do not aspirate after incubation
Add 20 μl 1x streptavidin-PE.
Incubate at 850 ± 50 rpm for 30 min at RT
Wash 3x with 100 µl assay buffer (1x)
Resuspend beads in 100 μl assay buffer (1x).
Shake at 850 ± 50 rpm for 30 sec
Read plate on Bio-Plex system
6
lmportant Considerations
Instruments and Software
The 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.
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 comes complete with a printed Bio-Plex Pro RBM Kidney
Toxicity Assays quick guide (bulletin #10028259), which can be used to
set up and prepare a full 1 x 96-well assay plate. Users can also download
a copy at www.bio-rad.com/bio-plex.
Bead Regions and Multiplexing Compatibility
n
Bead regions for all analytes are listed in the Read Plate section
n
Do not mix analytes between different kidney toxicity panels, or with
other Bio-Plex assay panels or reagents
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
Determine the total number of wells in the experiment using the Plate Layout
Template on page 28 or the Plate Formatting tab in Bio-Plex Manager™
software. A suggested plate layout 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
standard diluent alone and be processed in the same manner as
sample and standard wells. Note that Bio-Plex Manager automatically
subtracts the blank (B) MFI value from all other assay wells.
3.User-specific controls, as well as the quality controls supplied in the
kits, are assigned to wells in columns 3 and 4.
4. The remainder of the plate is available for samples.
Legend
S
Standards
B
Blank
X
Samples
C
Controls
Fig. 2. Suggested plate layout. For detailed instructions on
plate formatting in Bio-Plex Manager, see section Read Plate.
8
2. Prepare Instrument
These directions are specific for the Bio-Plex® 100/200 reader. To prepare
either a Bio-Plex 3D or Bio-Plex® MAGPIX™ reader, consult their respective
user manuals.
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.
Note: While the instrument is warming up, bring the 10x assay buffer and
sample dilution buffer to room temperature. Keep other items on ice until
needed. Also, begin to thaw frozen samples.
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 (Bio-Plex 100, 200, or similar).
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.
9
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 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
Setting up the Bio-Plex Pro or Bio-Plex Pro II
Wash Station
The wash station does not require calibration; however, it should be primed
before use. For more information, refer to the Bio-Plex Pro and Pro II wash
station quick guide (bulletin #5826).
1.Install the appropriate plate carrier on the wash station.
2. Use the prime procedure to prime channel 1 with wash buffer.
Setting Up the Bio-Plex Handheld Magnetic Washer
Place an empty flat bottom plate on the magnetic washer by sliding
it under the retaining clips. Push the clips inward to secure the plate.
Make sure the plate is held securely. If needed, the clips can be adjusted
for height and tension. For detailed instructions, refer to the user guide
(bulletin #10023087).
10
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 Reagents
1. Reconstitute the following lyophilized reagents in dH20 before use
according to the table below.
Table 4. Reagent volume.
Reagent
dH2O Volume
Standards mix
150 µl
Control 1
100 µl
Control 2
100 µl
Blocking buffer
1.5 ml
Standard diluent
1.0 ml
Detection antibodies
4.8 ml
a. Allow vial to sit at room temperature for a minimum of 5 min, not to
exceed 30 min.
b. Mix by vortexing at a medium setting.
2. Bring the 10x assay buffer to room temperature (RT).
a. Mix by inversion to ensure all salts are into solution.
b. P
repare 1x assay buffer — dilute 1 part 10x assay buffer with
9 parts of dH20.
11
Dilution of Standard (1:3 Serial Dilution)
This procedure provides sufficient volume to run duplicate standard
dilution curves. Ensure that each new standard is mixed well by vortexing
before proceeding to the next dilution. Change tips between each dilution.
Note: The product data sheet in each kit lists the most concentrated
point on the standard curve (S1). Enter the values and units into Bio-Plex
Manager™ software as instructed in the Read Plate section.
1. Label 8 polypropylene tubes S1 through S8. Label an additional
tube Blank.
2. Transfer the reconstituted standard into the tube labeled “S1.”
3. Add the appropriate amount of the standard diluent into the labeled
tubes according to Figure 3 (this will be sufficient for duplicate
standard curves and blanks).
4. Prepare working standards (S2–S8) by 1:3 (threefold) serial dilution.
Transfer the appropriate volume of standard into each of the labeled
tubes containing standard diluent.
5. Vortex each standard at a medium setting for 5 sec before proceeding
with the next serial dilution. Change pipet tip at each dilution step.
15050505050505050
Reconstituted Standard
Transfer Volume, µl
100100100100100100100100 Standard Diluent, µl
S1S2S3S4S5S6S7S8
Blank
Fig. 3. Preparing a threefold dilution series with a single reconstituted standard.
12
5. Prepare Samples
General guidelines for preparing urine samples are provided here. For more
information contact Bio-Rad Technical Support.
1. Centrifuge urine samples at 500 x g for 5 min to remove particulates.
2. Transfer the cleared samples into new aliquot tubes.
3. Place on ice for immediate use, or store as single-use aliquots at –70 ˚C.
Avoid multiple freeze-thaws.
4. Thaw frozen samples completely prior to dilution.
5. Within 1 hr after thawing, prepare sample dilutions in polypropylene tubes
(such as Titertube® micro test tubes) as recommended in Table 5. Dilution
scenarios provided are sufficient to run each sample in duplicate.
6. Equilibrate diluted samples to room temperature just prior to the start of
the assay. Do not freeze diluted samples.
Table 5. Summary of recommended sample dilutions.
Panel
Sample Dilution
Volume of
Urine Sample, µl
Volume of
Sample Buffer, µl
Human Tox 1
1:4
20
60
Human Tox 2
1:50
10
490
Rat Tox 1
1:2
40
40
Rat Tox 2
1:50
10
490
Rat Albumin
1:10,000
Canine Tox 1
1:15
Canine Albumin
1:10,000
5 (A. Prepare 1:100) 5 (B. P
repare 1:100)
10
5 (C. Prepare 1:100)
5 (D. P
repare 1:100)
Note: Controls are ready to use after reconstitution. No dilution is needed.
13
495
495
140
495
495
6. Run the Assay
Considerations
n
n
n
n
Bring all assay components and samples to room temperature before use
Use calibrated pipets and pipet carefully, avoiding bubbles. Use new
pipet tips for every volume transfer
Pay close attention to vortexing, shaking, and incubation instructions.
Deviation from the protocol may result in low assay signal and
assay variability
Assay incubations are carried out in the dark on a shaker at 850 ± 50 rpm.
Cover the plate with a plate seal and protect from light with aluminum foil
Table 6. Summary of wash options and protocols. 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
Sample incubation
MAG x3
VAC x3
SA-PE incubation
3 x 100 μl
Considerations When Using a Vacuum Manifold
n
n
n
n
n
After each incubation, place the filter plate on a calibrated vacuum
apparatus and remove the liquid by vacuum filtration
To wash, add 100 μl wash buffer to each well and remove the liquid as
before. Ensure that all wells are exposed to the vacuum
Thoroughly blot the bottom of the filter plate with a clean paper towel
between each vacuum step to prevent cross contamination
Place the assay plate on the plastic plate holder/tray as needed
Before each incubation, gently cover the plate with a new plate seal.
Avoid pressing down over the wells to prevent leaking from the bottom
14
Assay Protocol: Dispensing of Reagents
1. Add 10 µl of blocking buffer to all wells of the plate.
2. Add 30 µl of the standard, blank, control, or sample to the appropriate
well of the plate.
3. Vortex the capture beads at medium speed for 10–20 sec. Add 10 µl of
the beads to all wells of the plate.
4. Cover plate with plate seal and protect from light with aluminum foil.
Incubate on shaker at 850 ± 50 rpm for 1 hr at RT.
5. Wash the plate three times with 100 µl 1x assay buffer.
6. Vortex the reconstituted detection antibodies at medium speed for
10–20 sec. Add 40 µl to each well.
7. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 1 hr at RT.
8. Prepare the required dilution of streptavidin-PE (SA-PE) as outlined in
Table 7.
Note: Volumes in the table are for an entire 96-well plate. Smaller
volumes can be prepared, provided that dilution ratios are maintained.
Table 7. SA-PE dilution.
SA-PE Dilution
Volume of
SA-PE, µl
Volume of
1x Assay Buffer, µl
Total Volume, µl
1:10
225
2,025
2,250
9. Add 20 µl of diluted SA-PE to the required plate wells.
10. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 30 min at RT.
11. Wash the plate three times with 100 µl 1x assay buffer.
12. After the final wash, resuspend the beads in 100 µl assay buffer. Cover
plate as in Step 4 and shake the plate at 850 ± 50 rpm for 30 sec.
13. Remove the plate seal and read plate at low PMT (Bio-Plex® 200),
standard PMT (Bio-Plex 3D), or default settings (Bio-Plex® MAGPIX™).
15
7. 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 used in the reading, the plate wells
to be read, sample information, the values of standards and controls,
and instrument settings. Protocols may be obtained from within Bio-Plex
Manager software version 6.0 and higher or created from the File menu.
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 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
in the Select Analytes toolbar. Enter a new panel name. Select Bio-Plex Pro Assay Magnetic
from the assay dropdown menu. If using Bio-Plex Manager version 5.0 or lower, select MagPlex from the assay dropdown list.
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 for the individual assays are listed
in Table 8.
16
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.
Table 8. Bead regions for Bio-Plex Pro RBM kidney toxicity panels.
Human Kidney Bead
Tox Panel 1 Region
Human Kidney Bead
Tox Panel 2
Region
Calbindin
Clusterin
GST-p
IL-18
KIM-1
MCP-1
Albumin
B2M
Cystatin C
NGAL
Osteopontin
TFF3
30
22
51
46
20
61
Canine
Albumin Kit
Bead
Region
64
12
25
21
44
15
Canine Kidney Bead
Tox Panel 1 Region
Clusterin
KIM-1
MCP-1
NGAL
12
Albumin
53
15
46
30
Rat Kidney Tox Panel 1 Bead
Region
Bead
Region
Clusterin
IL-18
KIM-1
MCP-1
Osteopontin
12
B2M
22
Albumin 21
Calbindin
62
20
Cystatin C
44
15
NGAL
46
52
Rat Kidney
Tox Panel 2
17
Rat Albumin
Kit
Bead
Region
30
3.Click Format Plate and format the plate 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 .
Fig. 4. Plate formatting.
4. Click Enter Standards Info in the Protocol Settings bar.
a.Enter the highest concentration 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 3 and click Calculate. The concentrations
for each standard point will be populated for all analytes in the table.
18
c. Optional: enter the lot number of the vial of standards into the
Standard Lot box and click Save.
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 kit 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 “quality 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 9.
Table 9. 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, 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.
19
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 plate seal and any plate cover before placing
the plate on the plate carrier.
2.Click Run Protocol and 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
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. 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.
20
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 can also 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.
21
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 3D 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, which
automatically sets the DD gates.
2. Volume = 50 µl
3. 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 “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.
22
Troubleshooting Guide
This troubleshooting guide addresses problems that may be encountered
with Bio-Plex Pro™ RBM 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 assist in
determining if the array reader is functioning properly.
Possible Causes
Standards were not reconstituted
consistently between assays
Possible Solutions
Reconstituted standards 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.
High Inter-Assay CV
Incubate the reconstituted
standards for 30 min on ice. Always
be consistent with the incubation
time and temperature.
23
Possible Causes
Possible Solutions
High Intra-Assay CV
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.
Reagents and assay components
not equilibrated to room
temperature prior to pipetting
All reagents and assay components
should be equilibrated to room
temperature prior to pipetting.
Contamination with buffer
during wash steps
During the wash steps, be careful
not to splash 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.
Improper pipetting technique
Low Bead Count
Beads clumped in multiplex
bead stock tube
Vortex for 30 sec at medium speed
before aliquoting beads.
24
Possible Causes
Possible Solutions
Low Bead Count
Vacuum on for too long when
aspirating buffer from wells
Do not apply vacuum to the filter
plate for longer than 10 sec after the
buffer is completely drained from
each well.
Reader is clogged
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 standard preparation
instructions carefully.
Detection antibody or streptavidin-PE diluted incorrectly
Check your calculations and be
careful to add the correct volumes.
Incorrect buffer was used
(for example, assay buffer used
to dilute standards)
High Background Signal
Accidentally spiked blank wells
Detection antibodies or
streptavidin-PE incubated too long
Use standard diluent to dilute
standards.
Do not add any antigens to the
blank wells.
Follow the procedure incubation
time precisely.
25
Possible Causes
Possible Solutions
Poor Recovery
Expired Bio-Plex reagents
were used
Check that reagents have not
expired. Use new or nonexpired
components.
Incorrect amounts of components
were added
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.
Controls do not fall within
expected ranges
Make sure that the vial of controls
is reconstituted at the same time as
standards and in the correct diluent.
Incubate for times indicated.
Improper pipetting technique
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.
26
Possible Causes
Possible Solutions
Impact of Sample Matrix
Negative MFI values in samples
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 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.
27
Plate Layout Template
28
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
(U.S. government publication: Biosafety in Microbiological and Biomedical
Laboratories (CDC, 1999)).
Legal Notices
Bio-Plex Pro RBM kits are manufactured by Myriad RBM.
Acrodisc and Supor are trademarks of Pall Corporation. MagPlex, xMAP,
xPONENT, FLEXMAP 3D, and Luminex are trademarks of Luminex
Corporation. Myriad RBM is a trademark of Myriad RBM, Inc.
The Bio-Plex suspension array system includes fluorescently labeled
microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc.
by the Luminex Corporation.
29
Ordering Information
Detailed ordering information can be found at www.bio-rad.com/bio-plex.
Catalog #
Premixed All-In-One Multiplex Kit
Includes premixed magnetic capture beads, premixed detection antibodies, standards mix, 2-level controls,
blocking buffer, standard diluent, sample dilution buffer, 10x assay buffer, 10x streptavidin-PE, 96-well flat
bottom plate, plate seals, and instructions.
171-ATR1CK
171-ATR2CK
171-KTR1CK
171-KTR2CK
171-KTR3CK
171-QTR1CK
171-QTR2CK
Bio-Plex Pro RBM Human Kidney Toxicity Panel 1, 1 x 96
Bio-Plex Pro RBM Human Kidney Toxicity Panel 2, 1 x 96
Bio-Plex Pro RBM Rat Kidney Toxicity Panel 1, 1 x 96
Bio-Plex Pro RBM Rat Kidney Toxicity Panel 2, 1 x 96
Bio-Plex Pro RBM Rat Kidney Toxicity Albumin Kit, 1 x 96
Bio-Plex Pro RBM Canine Kidney Toxicity Panel 1, 1 x 96
Bio-Plex Pro RBM Canine Kidney Toxicity Albumin Kit, 1 x 96
Catalog #
Wash Stations and Accessories
300-34376
Bio-Plex Pro Wash Station, includes magnetic plate carrier, waste bottle, 2 buffer bottles
300-34377
Bio-Plex Pro II Wash Station, includes magnetic plate carrier, vacuum manifold plate
carrier, waste bottle, 2 buffer bottles
171-020100
Bio-Plex Handheld Magnetic Washer, includes magnetic washer and adjustment hex
tools for use in manual wash steps for all Bio-Plex magnetic assays
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
using the vacuum wash method, sealing tape not included
Catalog #
Software
171-001510
Bio-Plex Data Pro with Bio-Plex Manager Software (5 seats), for multi-experiment
analysis and advanced data visualization
171-001513
Bio-Plex Data Pro Software (5 seats), for multi-experiment analysis and advanced data
visualization
171-001523
Bio-Plex Data Pro Plus Software, contains all the features of Bio-Plex Data Pro
software with added visualization, sharing, and analysis functionality
171-STND01
Bio-Plex Manager Software (1 seat), for instrument data evaluation and optimization
30
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