Bio-Plex Pro™ RBM IGF and IGFBP Assays - Bio-Rad

Bio-Plex Pro™ RBM IGF and IGFBP Assays - Bio-Rad
Bio-Plex Pro RBM
IGF and IGFBP 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
Principle
3
Kit Contents and Storage
5
Recommended Materials
6
Assay Workflow
7
lmportant Considerations
8
Detailed Instructions 8
1. Plan Plate Layout
9
2. Prepare Instrument
10
3. Prepare Wash Method11
4. Prepare Reagents12
5. Prepare Samples14
6. Run the Assay18
7. Read Plate
20
Troubleshooting Guide
26
Plate Layout Template
31
Safety Considerations
32
Legal Notices
32
Ordering Information
33
Introduction
Insulin-like growth factors (IGFs), along with their binding proteins (IGFBPs),
have been shown to play key roles in cell growth, organogenesis, and in the
pathophysiology of various cancers. Levels of IGF-1 gradually increase after
birth, peak at puberty, and decline thereafter. Unlike insulin, IGFs circulate
in biological fluids, mostly complexed to IGFBPs. IGF-2 is typically low at
birth and increases with age until puberty, then levels remain unchanged
throughout the rest of life. Serum IGF-2 is roughly twice that of IGF-1
(Meinbach and Lokeshwar 2006). IGFs are regulated by IGFBPs, which bind
the free IGFs to form a ternary complex and modulate IGF binding to the
IGF receptors (IGFRs) on endothelium.
The IGFBPs are divided into high affinity IGFBPs (IGFBP-1 through IGFBP-6)
and low affinity IGFBPs (IGFBP-7 through IGFBP-10). IGFBP-1 through
IGFBP-6 have been well characterized, and are capable of binding both
IGF-1 and IGF-2 with higher affinity than the IGF-IGFR interaction (Juul
2003). Of all the IGFBPs, IGFBP-1 is known to be nutritionally regulated,
with serum levels highest during fasting and lowest postprandial. IGFBP-3
is the most abundant and binds as much as 99% of the circulating IGF
pool (Hjortebjerg and Frystyk 2013). IGFBP-2 is the second most abundant
and, like IGFBP-1, links the IGF system with insulin sensitivity, glucose
metabolism, and cancer. IGFBP-4 is mostly derived from the liver. Its serum
concentration represents less than 10% of IGFBP-3 serum concentration.
Serum levels of IGFBP-4 and IGFBP-6 were found to increase with age
while the opposite was found with IGFBP-5. IGFBP-7 is expressed in a
variety of tissues including heart, spleen, ovary, and intestine. Unlike the
other six members, it has roughly 100-fold lower affinity for IGF-1 while its
affinity for insulin is 500-fold higher (Kutsukake 2008).
The usefulness of serum IGF-1, IGFBP-3, and serum IGF-1/IGFBP-3 molar
ratio is well documented in the diagnosis and treatment of growth and
metabolic disorders, diabetes, cardiovascular diseases, and cancers.
1
Bio-Plex Pro™ RBM IGF and IGFBP Assays
The Bio-Plex Pro RBM IGF and IGFBP assays include a wide range of
biomarkers 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
Hjortebjerg R and Frystyk J (2013). Determination of IGFs and their
binding proteins. Best Pract Res Clin Endocrinol Metab 27, 771–781.
Juul A (2003). Serum levels of insulin-like growth factor 1 and its binding
proteins in health and disease. Growth Horm IGF Res 13, 113–170.
Kutsukake M. et al. (2008). Circulating IGF-binding protein 7 (IGFBP7)
levels are elevated in patients with endometriosis or undergoing diabetic
hemodialysis. Reprod Biol Endocrinol 6, 54.
Meinbach DS and Lokeshwar BL (2006). Insulin-like growth factors and
their binding proteins in prostate cancer: cause or consequence? Urol
Oncol 24, 294–306.
2
Principle
Technology
The Bio-Plex® multiplex system is built upon the three core elements of
xMAP technology:
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
n
Assay Format
The Bio-Plex Pro™ RBM IGF and IGFBP 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.
3
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. The concentration of analyte
bound to each bead is proportional to the MFI of reporter signal.
4
Kit Contents and Storage
The Bio-Plex Pro™ RBM IGF and IGFBP 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.
Component
Quantity
Volume
Volume after
Reconstitution or Dilution
Capture beads (1x)
1 tube
Detection antibodies
1 vial
Lyophilized
1.4 ml
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 2
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 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.
5
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
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).
6
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
Do not aspirate after incubation
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
7
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 IGF
and IGFBP Assays Quick Guide (bulletin #10042014), 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
Do not mix analytes between different panels, or with other Bio-Plex
assay panels or reagents
n
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.
8
1. Plan Plate Layout
Determine the total number of wells in the experiment using the Plate
Layout Template on page 31 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.
9
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.
10
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.
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).
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
1.Install the appropriate plate carrier on the wash station.
2. Use the prime procedure to prime channel 1 with 1x assay buffer.
11
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).
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).
12
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.
15050505050505050
Reconstituted Standard
Transfer Volume, µl
100100100100100100100100 Standard Diluent, µl
S1S2S3S4S5S6S7S8
Blank
Fig. 3. Preparing a threefold dilution series with a single reconstituted standard.
13
5. Prepare Samples
Note: Most of the circulating IGF-1 and IGF-2 are complexed with the
IGFBPs. An extraction step to separate the complexes is required prior
to measuring the IGFs in serum and plasma samples. Users measuring
free IGF should skip the sample extraction step and proceed to dilute
the sample 1:5 with the provided sample dilution buffer. Thaw and dilute
samples within 1 hr of use. Remove any particulates by centrifugation or
filtration. Avoid multiple freeze and thaw cycles.
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.
Note: Plasma is preferred for detection of IGFBP-5, as levels may be below
detection in serum.
n
n
Avoid using hemolyzed samples, as this may lead to false results.
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
14
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 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.
15
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 stablize protein analytes and to prevent adsorption to labware.
2. Transfer to a clean polypropylene tube. If cellular debris or precipitates are present, centrifuge 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. Assay 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.
Sample Extraction for IGF-1 and IGF-2
Prepare fresh extraction and neutralization buffers immediately before use.
1. For each sample label two 1.5 ml polypropylene microcentrifuge tubes, one for extraction and one for neutralization.
2. Pipet 20 µl sample into the corresponding 1.5 ml extraction tube.
3. Add 180 µl extraction buffer (12.5% 2 N HCl and 87.5% ethanol) to each extraction tube for a 1:10 dilution.
Table 5. Preparation of IGF extraction buffer.
Materials
5N HCI
DI water, 18 mΩ Super-Q purified
Ethyl alcohol, denatured
Vendor/Part #
Amount required (ml)
Fisher/NC9053751
0.500
N/A
0.7500
Fisher/50-980-458
8.750
4.Cap and vortex each tube. Incubate for 30 min (±2 min) at RT. After
the initial 15 min incubation, vortex the tubes and allow to sit for the
remaining 15 min.
16
5. After the incubation, centrifuge the extraction tubes in a microcentrifuge at 8,400 x g for 10 min (±1 min).
6.Add 50 µl neutralization buffer (1 M Tris, pH 7.5) to each of the tubes
labeled for neutralization.
Table 6. Preparation of IGF neutralization buffer.
Materials
1 M Tris-HCI, pH 7.5
Vendor/Part #
Amount required (ml)
Fisher/NC9053751
As needed
7. Before transferring the extract into neutralization buffer, prewet each
pipet tip by pipetting up and down 2–3 times in a small amount
(~1 ml) of extraction buffer. This should be done in a clean 1.5 ml tube.
Note: Do not prewet the pipet tip in the extracted sample, as this may resuspend a portion of the compacted pellet.
8. Without disturbing the pellet, transfer 50 µl extracted supernatant
to the corresponding neutralization tube (containing 50 µl of
neutralization buffer), cap and, vortex.
9. Add 50 µl sample dilution buffer 2 to the neutralized sample. Final sample dilution is 1:30.
10.Store samples on ice for immediate testing. Alternatively, store them at –70°C for up to one month.
17
Table 7. Serum and plasma sample dilution guidelines.
Panel
IGF
IGFBP
Sample
Dilution
Volume of
Sample, µl
Volume of
Extraction
Buffer
1:30
20
180 µl
1:20
10
N/A
Volume of
Neutralization
Buffer
Volume of Sample
Dilution Buffer 2
Mix 50 µl extracted
Mix the neutralized
sample with
sample with
50 µl neutralization
50 µl sample dilution
buffer
buffer 2
N/A
190 µl
Note: Sample dilution factor for culture media samples must be optimized
by the end user.
6. Run the Assay
Considerations
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
n
Table 8. 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
VAC x3
3 x 100 μl
After SA-PE incubation
MAG x3
18
Considerations When Using a Vacuum Manifold
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
n
Assay Protocol: Dispensing of Reagents
1.Add 10 µl blocker to all wells of the plate.
2.Add 30 µl 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
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. Do not aspirate
after incubation.
5.Vortex the reconstituted detection antibodies at medium speed for
10–20 sec. Add 40 µl to each well.
6.
Cover and incubate at 850 ± 50 rpm, as in Step 4, for 1 hr at RT. Do
not aspirate after incubation.
7.
Prepare streptavidin-PE (SA-PE) as outlined in Table 9.
Note: Volumes in the table are for an entire 96-well plate. Smaller
volumes can be prepared, provided that dilution ratios are maintained.
Table 9. SA-PE dilution.
SA-PE Dilution
1:10
Volume of
SA-PE, µl
Volume of
1x Assay Buffer, µl
Total Volume, µl
225
2,025
19
2,250
8.Add 20 µl diluted SA-PE to the required plate wells.
9.Cover and incubate at 850 ± 50 rpm, as in Step 4, for 30 min at RT.
10.Wash the plate three times with 100 µl 1x assay buffer.
11.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.
12.Remove the plate seal and read plate at low PMT (Bio-Plex® 200),
standard PMT (Bio-Plex 3D), or default settings (Bio-Plex® MAGPIX™).
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.
20
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 10.
c. Click Add 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 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 10. Bead regions for Bio-Plex Pro RBM IGF and IGFBP assays.
IGF Panel
Bead Region
IGF BP Panel
Bead Region
IGF-1
21
IGFBP-1
48
IGF-2
54
IGFBP-2
65
IGFBP-3
33
IGFBP-4
76
IGFBP-5
51
IGFBP-6
78
IGFBP-7
37
21
3.Click Format Plate and format the plate according to the plate layout
template (located on page 31) created for the assay. 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. E
nter 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.
22
5.Click Enter Controls Info.
a. F
or 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. F
or 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 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,
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.
23
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
the instructions.
and follow
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.
24
Previous Versions of Bio-Plex Manager Software
For instructions on using previous versions of Bio-Plex Manager software,
please contact Bio-Rad Technical Support.
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.
25
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
Possible Solutions
Reconstituted standards and
diluted samples were not stored
properly
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.
Incubate the reconstituted standards
for for the recommended time
period. Always be consistent with the
incubation time and temperature.
26
Possible Causes
Possible Solutions
High Intra-Assay CV
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.
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.
Low Bead Count
Beads clumped in multiplex
bead stock tube
Vortex for 30 sec at medium speed
before aliquoting beads.
27
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 prepared incorrectly
Check your calculations and be
careful to add the correct volumes.
High Background Signal
Incorrect buffer was used
(for example, assay buffer used
to dilute standards)
Use standard diluent to dilute
standards.
Accidentally spiked blank wells
Do not add any antigens to the
blank wells.
Detection antibodies or
streptavidin-PE incubated too long
Follow the procedure incubation
time precisely.
28
Possible Causes
Poor Recovery
Expired Bio-Plex reagents
were used
Possible Solutions
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.
Check that reagents have not
expired. Use new or nonexpired
components.
29
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)
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.
30
Plate Layout Template
31
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. Super-Q is a trademark of Merck KGaA
Darmstadt Germany. 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.
32
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 2, 10x assay buffer, 10x streptavidin-PE, 96-well flat
bottom plate, plate seals, and instructions.
171-AGR1CK
171-AFR1CK
Bio-Plex Pro RBM Human IGFBP Panel, 1 x 96
Bio-Plex Pro RBM Human IGF Panel, 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
33
Bio-Rad
Laboratories, Inc.
Web site www.bio-rad.com USA 800 424 6723
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Greece 30 210 9532 220 Hong Kong 852 2789 3300
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Italy 39 02 216091 Japan 81 3 6361 7000 Korea 82 2 3473 4460
Mexico 52 555 488 7670 The Netherlands 0318 540666
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Taiwan 886 2 2578 7189 Thailand 1800 88 22 88
United Kingdom 020 8328 2000
Life Science
Group
10042015 Rev A
US/EG
Sig 1213
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