Bio-Plex Pro
™
RBM
Metabolic and Hormone
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
Introduction 1
Principle 2
Kit Contents and Storage 4
Recommended Materials 5
Assay Workflow 6
lmportant Considerations 7
Detailed Instructions 7
1. Plan Plate Layout 8
2. Prepare Instrument 9
3. Prepare Wash Method 10
4. Prepare Reagents 11
5. Prepare Samples 13
6. Run the Assay 16
7. Read Plate 18
Troubleshooting Guide 24
Plate Layout Template 29
Safety Considerations 30
Legal Notices 30
Ordering Information 31
Introduction
Approximately 65% of individuals with diabetes die from heart disease
and stroke. Individuals with type 2 diabetes also have an increased rate of
high blood pressure, lipid problems, and obesity, which contribute to their
high rates of cardiovascular disease (CVD). Given the complexity of these
conditions, it is not surprising that assessment of a single biomarker alone has
not yielded more than an incremental advantage over traditional measures
of assessing risk. In diabetes, further complexity is added by the common
coincidence of metabolic syndrome, which includes insulin resistance,
dyslipidemia, hypertension, and systemic inflammation, all of which contribute
to the pathogenesis of CVD (Iqbal et al. 2012). As such, researchers studying
CVD and/or diabetes need to monitor multiple biomarkers for cardiovascular
risk factors along with biomarkers directly linked to the metabolic disorders
mentioned above. These markers include gut and pituitary hormones,
adipokines, growth factors, and cardiovascular and acute phase–related
proteins. Since these hormones and proteins frequently act in concert, it
is advantageous to have the capacity to measure a number of different
biomarkers simultaneously in a single blood sample.
Bio-Plex Pro™ RBM Metabolic and Hormone Assays
The Bio-Plex Pro RBM metabolic and hormone assays include a wide
range of proteins implicated in the pathophysiology of diabetes, obesity,
and metabolic syndrome, as well as biomarkers for associated conditions
such as cardiovascular disease and inflammation.
For researchers working with limited sample volume, the capacity to
multiplex provides an effective option over the traditional ELISA method.
The use of magnetic (MagPlex) beads allows researchers to automate
wash steps on a Bio-Plex Pro (or similar) wash station.
References
Iqbal N et al. (2012). Cardiac biomarkers: New tools for heart failure
management. Cardiovas Diagn Ther 2, 147–164.
1
Principle
Technology
The Bio-Plex® multiplex system is built upon the three core elements of
xMAP technology:
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 molecules in a single well of a 96-well
microplate on the Bio-Plex® 3D system, up to 100 different molecules
on the Bio-Plex® 200 system, and up to 50 different molecules on the
Bio-Plex® MAGPIX™ system
n
A dedicated plate reader. The Bio-Plex 200 and Bio-Plex 3D systems
are flow cytometry–based instruments 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
The Bio-Plex Pro™ RBM metabolic and hormone 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. 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.
Note: The cortisol assay in Metabolic Panel 1 is a competitive
immunoassay (schematic not shown).
2
Biomarker
of Interest
Streptavidin
Magnetic Bead
Capture
Antibody
Fig. 1. Bio-Plex sandwich immunoassay.
Biotinylated
Detection
Antibody
Phycoerythrin
Fluorescent
Reporter
Data Acquisition and Analysis
Data from the reactions are acquired using a Bio-Plex system or similar
Luminex-based reader. When a multiplex assay suspension is drawn into
the Bio-Plex 200 reader, for example, a red (635 nm) laser illuminates the
fluorescent dyes within each bead to provide bead classification and thus
assay identification. At the same time, a green (532 nm) laser excites PE
to generate a reporter signal, which is detected by a photomultiplier tube
(PMT). A high-speed digital processor manages data output and
Bio-Plex Manager™ software presents data as median fluorescence
intensity (MFI) as well as concentration. The concentration of analyte
bound to each bead is proportional to the MFI of reporter signal.
3
Kit Contents and Storage
The Bio-Plex Pro™ RBM metabolic and hormone assays are available
in a convenient kit format that includes assay, reagent, and diluent
components in a single box (Table 1). All other recommended materials
are listed in Table 2.
Table 1. Contents of 1 x 96-well kits.
Volume after
Component Quantity Volume Reconstitution or Dilution
Capture beads (1x) 1 tube 1.4 ml
Detection antibodies 1 vial Lyophilized 4.8 ml
Standards mix 1 vial Lyophilized 150 µl
Control 1 (high) 1 vial Lyophilized 100 μl
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 100 ml
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
Storage and Stability
Kit contents should be stored at 2–8°C and never frozen. Coupled
magnetic capture 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
Tab l e 2. Recommended materials.
Item
Bio-Plex® 200 system or Luminex system with HTF
Bio-Plex validation kit
Note: Run the validation kit monthly to ensure optimal
performance of fluidics and optics systems
Bio-Plex calibration kit
Note: Run the calibration kit daily to standardize
fluorescence signal
Bio-Plex Pro wash station
For use with magnetic bead–based assays only
Bio-Plex Pro II wash station
For use with both polystyrene (nonmagnetic) and magnetic
bead–based assays
Bio-Plex handheld magnetic washer
For use with magnetic bead–based assays only
Bio-Plex Pro flat bottom plates (40 x 96-well)
For magnetic separation on the Bio-Plex Pro wash station
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)
Aurum™ vacuum manifold
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
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).
Ordering Information
Bio-Rad catalog #171-000205
Bio-Rad catalog #171-203001
Bio-Rad catalog #171-203060
Bio-Rad catalog #300-34376
Bio-Rad catalog #300-34377
Bio-Rad catalog #170-20100
Bio-Rad catalog #171-025001
Bio-Rad catalog #223-9390
IKA catalog #320-8000
VWR catalog #57019-600
Bio-Rad catalog #732-6470
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
5
Assay Workflow
Prepare samples, reconstitute lyophilized
reagents, dilute assay buffer to 1x,
prepare standards
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.
Shake at 850 ± 50 rpm for 1 hr at RT
Wash 3x with 100 µl assay buffer (1x)
Add 40 μl reconstituted detection antibodies.
Shake at 850 ± 50 rpm for 1 hr at RT
Do not aspirate after incubation
Add 20 μl 1x streptavidin-PE.
Shake 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
Metabolic and Hormone Assays Quick Guide (bulletin #10041817), 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 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 29 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. Bio-Plex Manager software automatically
subtracts the assay 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.
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 Flat bottom plate
Bio-Plex Pro II (use MAG programs)
Bio-Plex® handheld magnetic washer
Vacuum filtration Bio-Plex Pro II (use VAC programs) Filter plate
Vacuum manifold (manual)
Setting Up the Bio-Plex Pro or Bio-Plex Pro II
Wash Station
The wash station should be primed before use. For more information, refer
to the Bio-Plex Pro Wash Stations Quick Guide (bulletin #5826).
1. Install the appropriate plate carrier on the wash station.
2. Use the prime procedure to prime channel 1 with 1x assay 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 in solution.
b. Prepare 1x assay buffer — dilute 1 part 10x assay buffer (60 ml) with
9 parts of dH20 (540 ml).
11
Dilution of Standard (1:3 Serial Dilution)
This preparation 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 9 polypropylene tubes S1 through S8 and 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 as outlined above.
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.
6. The Blank tube consists of standard diluent alone.
150 50 50 50 50 50 50 50
Transfer Volume, µl
Reconstituted
Standard
Fig. 3. Preparing a threefold dilution series with a single reconstituted standard.
100 100 100 100 100 100 100 100
S1 S2 S3 S4 S5 S6 S7 S8 Blank
Standard Diluent, µl
12
5. Prepare Samples
The kit has sufficient reagents to run 37 samples in duplicate for a full plate
analysis. General guidelines for preparing samples are provided below. For
more information, contact Bio-Rad Technical Support.
Serum and Plasma
Serum, EDTA plasma, or citrate plasma are the preferred sample types for
these assays. Heparin-treated plasma, while compatible with these assays,
may absorb certain soluble proteins of interest.
n Avoid using hemolyzed samples, as this may lead to false results.
n Freezing samples at –70°C immediately after preparation and keeping
samples frozen until use should provide adequate protection from
degradation. Avoid multiple freeze and thaw cycles.
n Once thawed, keep samples on ice.
n Do not freeze diluted samples
13
Protease Inhibitors
In general, these biomarkers are detectable in serum or plasma without
using protease inhibitors. Users may choose to add protease inhibitors as
a precautionary measure if samples will not be frozen immediately after
collection. Alternatively, blood sample collection tubes containing inhibitors
may be obtained from Becton-Dickinson, www.bd.com.
Note: Protease inhibitors are not recommended for use with serum
samples. Compatibility of a given inhibitor cocktail with the assays must
be validated by the end user.
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 30–45 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. Prepare sample dilutions in 0.5 or 1.0 ml polypropylene tubes as
required for the assay.
6. Dilution scenarios provided below are sufficient to run each sample in
duplicate.
14
Cell culture media
1. Collect cell culture supernatants and centrifuge at 1,000 x g for
15 min at 4°C. For cell lines cultured in serum-free media, collect
samples and add BSA as a carrier protein to a final concentration of
at least 0.5%. This is done 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. If high levels of analyte are expected, samples can be diluted in culture
media. Optimal dilution factor must be determined by the end user.
Supplement serum-free media with at least 0.5% BSA final.
4. Assays samples immediately or aliquot and store at –70°C.
Note: Compatibility of a given media type with the assays must be
validated by the end user.
Table 5. Serum and plasma sample dilution guidelines.
Panel Sample
Dilution
Metabolic panel 1 1:5 20 80
Metabolic panel 2 1:5 20 80
Metabolic panel 3 1:500,000 (a) Prepare 1:50 5
Metabolic panel 4 1:500 (c) Prepare 1:10 10
Hormone panel 1 1:5 20 80
Volume of Sample, µl Volume of
(b) Prepare 1:100 5 of (a)
Prepare 1:100 5 of (b)
Prepare 1:50 10 of (c)
Sample
Buffer, µl
245
495
495
90
490
Note: Sample dilution factor for culture media samples must be optimized
by the end user.
15
6. Run the Assay
Considerations
n
Bring all assay components and samples to room temperature before use
n
Use calibrated pipets and pipet carefully, avoiding bubbles. Use new
pipet tips for every volume transfer
n
Pay close attention to vortexing, shaking, and incubation instructions.
Deviation from protocol may result in low assay signal and assay variability
n
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, sele ct the
appropriate Bio-Plex Pro wash station program or perform the appropriate manual wash
step as summarized below.
Bio-Plex Pro or Bio-Plex Pro II Handheld Magnet or
Pro II Wash Station Wash Station Vacuum Manifold
Assay Step Magnetic Program Vacuum Program Manual Wash Steps
Sample incubation
MAG x3 VAC x3 3 x 100 μl
SA-PE incubation
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 μl 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 plate seal.
Avoid pressing down on the wells to prevent leaking from the bottom
16
Assay Protocol: Dispensing of Reagents
1. Add 10 µl blocker to all wells of the plate.
2. Add 30 µl the standard, 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. Do
not aspirate after incubation.
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.
Volume of Volume of
SA-PE Dilution SA-PE, µl 1x Assay Buffer, µl Total Volume, µl
1:10 225 2,025 2,250
9. Add 20 µl diluted SA-PE to the required plate wells.
10. Cover and incubate at 850 ± 50 rpm, as in Step 4, for 30 min a t 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™).
17
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 on 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 6.1 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.
a. Click Add Panel 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 menu.
b. Click Add. 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 the individual assays are listed in Table 8.
c. Click Add when the last analyte has been added and click OK to save
the new panel.
18
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 Add All.
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 metabolic and hormone assays.
Metabolic Panel 1 Bead Region Metabolic Panel 2 Bead Region
C-Peptide 45 FGF-21 63
Cortisol 12 FGF-23 47
Pancreatic polypeptide 52 Galectin-3 46
Proinsulin 67 sLeptin R 27
Peptide YY 55 Omentin-1 20
Pentraxin-3 44
PON-1 53
Vaspin 34
Metabolic Panel 3 Bead Region Metabolic Panel 4 Bead Region
AAT 26 ANGPTL3 66
AGP-1 57 Chemerin 42
Hemopexin 35 DPP4 25
RBP-4 43 Protein S 62
Transferrin 61 SEPP 36
Transthyretin 13
VDBP 28
Hormone Panel 1 Bead
Region
FSH 18
GH 38
LH 15
Prolactin 19
TSH 22
Note: The cortisol assay is a competitive immunoassay.
19
3. Click Format Plate and format the plate according to the plate layout
template (located on page 29) created for the assay. To modify the
plate layout, follow the steps below.
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.
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.
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 — enter sample information and the
appropriate dilution factor.
7. Click Run Protocol — confirm that the assay settings follow Table 9.
20
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
* A similar Luminex-based system may be used.
™
*
N/A, use default instrument settings
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.
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. Click 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 and follow
the instructions.
21
Data Analysis
Quality Controls
If the quality controls were run in the assay plate, open the results (.rbx)
file, click 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 (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 earlier versions of the software, 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.
22
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.
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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 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 were not reconstituted
consistently between assays
Reconstituted standards and
diluted samples were not stored
properly
Bottom of filter plate not dry
Possible Solutions
Incubate the reconstituted standards
for for the recommended time
period. Always be consistent with the
incubation time and temperature.
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.
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Possible Causes
High Intra-Assay CV
Improper pipetting technique
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.
Reagents and assay components
not equilibrated to room
temperature prior to pipetting
Contamination with buffer
during wash steps
Slow pipetting of samples and
reagents across the plate
Low Bead Count
Beads clumped in multiplex
bead stock tube
All reagents and assay components
should be equilibrated to room
temperature prior to pipetting.
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.
Sample pipetting across the entire
plate should take less than 4 min.
Reagent pipetting across the entire
plate should take less than 1 min.
Vortex for 30 sec at medium speed
before aliquoting beads.
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Possible Causes
Low Bead Count
Vacuum on for too long when
aspirating buffer from wells
Possible Solutions
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
Low Signal or Poor Sensitivity
Standards reconstituted
incorrectly
Detection antibody or
streptavidin-PE prepared incorrectly
High Background Signal
Incorrect buffer was used
(for example, assay buffer used
to dilute standards)
Accidentally spiked blank wells
Detection antibodies or
streptavidin-PE incubated too long
Refer to the troubleshooting guide
in the Bio-Plex system hardware
instruction manual (bulletin
#10005042).
Follow the standard preparation
instructions carefully.
Check your calculations and be
careful to add the correct volumes.
Use standard diluent to dilute
standards.
Do not add any antigens to the
blank wells.
Follow the procedure incubation
time precisely.
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Possible Causes
Poor Recovery
Expired Bio-Plex reagents
were used
Possible Solutions
Check that reagents have not
expired. Use new or nonexpired
components.
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
Improper pipetting technique
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 for data analysis
if needed.
Make sure that the vial of controls
is reconstituted at the same time as
standards and in the correct diluent.
Incubate for times indicated.
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.
2727
Possible Causes
Impact of Sample Matrix
Negative MFI values in samples
Possible Solutions
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)
MFI 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 MFI value if the blank’s
MFI 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.
2828
Plate Layout Template
29
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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. MAGPIX,
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.
30
30
Ordering Information
Detailed ordering information can be found at www.bio-rad.com/bio-plex.
Catalog # Premixed 1 x 96-Well 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-AMR1CK Bio-Plex Pro RBM Human Metabolic Panel 1, 1 x 96
171-AMR2CK Bio-Plex Pro RBM Human Metabolic Panel 2, 1 x 96
171-AMR3CK Bio-Plex Pro RBM Human Metabolic Panel 3, 1 x 96
171-AMR4CK Bio-Plex Pro RBM Human Metabolic Panel 4, 1 x 96
171-AHR1CK Bio-Plex Pro RBM Human Hormone Panel 1, 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
171-020100 Bio-Plex Handheld Magnetic Washer, includes magnetic washer and adjustment hex
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
Catalog # Software
171-001510 Bio-Plex Data Pro™ with Bio-Plex Manager Software (5 seats), for multi-experiment
171-001513 Bio-Plex Data Pro Software (5 seats), for multi-experiment analysis and advanced
171-001523 Bio-Plex Data Pro Plus Software, contains all the features of Bio-Plex Data Pro
171-STND01 Bio-Plex Manager Software (1 seat), for instrument data evaluation and optimization
carrier, waste bottle, 2 buffer bottles
tools for use in manual wash steps for all Bio-Plex magnetic assays
using the vacuum wash method, sealing tape not included
analysis and advanced data visualization
data visualization
software with added visualization, sharing, and analysis functionality
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Bio-Rad
Laboratories, Inc.
Life Science
Group
Web site ww w.bio-rad.com USA 800 424 6723
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Sig 121310041818 Rev A US/EG
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