10028370
Bio-Plex Pro Human
Isotyping Assays
™
Instruction Manual
For technical support, call your local Bio-Rad office, or in the U.S., call 1-800-424-6723.
For research use only. Not for diagnostic procedures.
Table of Contents
Introduction1
Principle2
Kit Contents and Storage
4
Recommended Materials
5
Assay Workflow
6
lmportant Considerations
7
Detailed Instructions 7
1.1. Plan Plate Layout8
2.2. Prepare Instrument9
3.3. Prepare Wash Method10
4.4. Prepare Wash Buffer11
5.5. Prepare Standards and Controls11
6. Prepare Samples14
7.7. Prepare Coupled Beads16
8.8. Run Assay17
9.9. Read Plate21
Troubleshooting Guide
28
Plate Layout Template
33
Calculation Worksheet
34
Safety Considerations
35
Legal Notices
35
Ordering Information
36
Introduction
Bio-Plex Pro™ Human Isotyping Assays
Bio-Plex Pro isotyping assays enable you to measure multiple
immunoglobulin isotypes in only 5 μl of sample. A detailed profile of the
immune response to infection, disease, vaccination, or drug therapy can
now be obtained in a single three hour experiment. Bio-Plex Pro isotyping
assays are multiplex bead-based assays designed to quantitate multiple
immunoglobulin isotypes in diverse matrices, including serum, plasma,
and tissue culture supernatants. These assays are optimized for the
Bio-Plex suspension array system, which utilizes xMAP technology. For
a brief overview of the assay protocol, see the Bio-Plex isotyping assay
workflow in this manual. The Bio-Plex suspension array system, which
incorporates novel technology using color-coded beads, allows the
simultaneous detection of up to 100 analytes in a single well of a 96-well
microplate. The advantages over traditional methods of immunoglobulin
isotyping include the ability to create a complete quantitative
immunoglobulin class and subclass profile from limited sample, reduced
sample preparation and assay time, and increased throughput. The use
of magnetic beads allows researchers to automate wash steps.
Advantages of Magnetic Bead-Based Assays
Products in the Bio-Plex Pro family of assays are on magnetic polystyrene
beads. These beads provide a choice in the method of assay preparation —
standard or magnet-based. The standard workflow for Bio-Plex assay
preparation requires multiple wash steps in which the 96-well filter plate
is placed on a vacuum manifold to draw the liquid through the bottom of
the filter plate. In contrast, magnet-based assay preparation permits
liquid removal from the top of the well and thus does not require a filter
plate or vacuum manifold. As a result, either an automated or manual
washer can be used. Bio-Rad offers both solutions with the automated
Bio-Plex Pro wash station and the manual Bio-Plex handheld magnetic
washer. Magnetic separation offers greater convenience and reproducibility
compared to vacuum filtration.
For a current listing of Bio-Plex assays, panels, and reagents, please visit
www.bio-rad.com/bio-plex.
1
Principle
Technology
The Bio-Plex® suspension array system is built upon the three core
elements of xMAP technology:
n
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Fluorescently dyed microspheres (also called beads), each with a distinct
color code or spectral address to permit discrimination of individual tests
within a multiplex suspension. This allows simultaneous detection of more
than 100 different types of molecules in a single well of a 96-well microplate
A dedicated flow cytometer with two lasers and associated optics to
measure the different molecules bound to the surface of the beads.
The Bio-Plex® MAGPIX™ uses LED and CCD technology to accomplish
the same
A high-speed digital signal processor that efficiently manages the fluorescence data
n
Assay Format
Bio-Plex Pro™ assays are essentially immunoassays formatted on
magnetic beads. The assay principle is similar to that of a sandwich ELISA
(Figure 1). Capture antibodies directed against the desired biomarker
are covalently coupled to the beads. Coupled beads react with the
sample containing the biomarker of interest. After a series of washes to
remove unbound protein, a biotinylated detection antibody is added to
create a sandwich complex. The final detection complex is formed with
the addition of streptavidin-phycoerythrin (streptavidin-PE or SA-PE)
conjugate. Phycoerythrin serves as a fluorescent indicator, or reporter.
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. The concentration of analyte bound to each bead is
proportional to the MFI of the reporter signal.
3
Kit Contents and Storage
Reagents Supplied
Bio-Plex Pro™ isotyping assays are available in a convenient all-in-one kit
format that includes assay, reagent, and diluent components in a single box.
Table 1. Contents of 1 x 96-well kits.
Component
Quantity*
Isotyping diluent
1 bottle, 180 ml
Assay buffer
1 bottle, 50 ml
Wash buffer (10x)
1 bottle, 60 ml
Detection antibody diluent
1 bottle, 5 ml
Streptavidin-PE (100x)
1 tube
Assay plate (96-well flat bottom plate)
1 plate
Sealing tape
Assay quick guide
Product data sheet
1 pack of 4
1 booklet
1
Coupled magnetic beads (20x)
1 tube
Detection antibodies (20x)
1 tube
Standard
1 vial
Quality Control
1 vial
* Volumes shown are approximate.
Storage and Stability
Kit contents should be stored at 4°C and never frozen. Coupled magnetic
beads and streptavidin-PE should be stored in the dark. All components
are guaranteed for a minimum of six months from the date of purchase
when stored as specified.
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 (forty 96-well plates)
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
Prewet wells
(for filter plate only)
Add 50 µl 1x beads to wells
Wash 2 x 100 μl
Add 50 μl diluted standards, samples, controls,
incubate 1 hr at RT with shaking at 850 rpm
Wash 3 x 100 μl
Add 25 μl 1x detection antibody, incubate
30 min at RT with shaking at 850 rpm
Wash 3 x 100 μl
Add 50 μl 1x streptavidin-PE, incubate
10 min at RT with shaking at 850 rpm
Wash 3 x 100 μl
Resuspend in 125 μl assay buffer,
shake at 850 rpm for 30 sec
Acquire data on Bio-Plex system
6
lmportant Considerations
Instruments and Software
The Bio-Plex Pro™ assays described in this manual are compatible with
all currently available Luminex-based life science research instruments.
Assays can be read and analyzed with either Bio-Plex Manager™ software
or Luminex xPonent software (section 9).
Assay Procedures
Pay close attention to vortexing, shaking, and incubation times and to
Bio-Plex® reader PMT (RP1) setting, as these have been optimized
specifically for each assay panel.
Assay Quick Guide
Each assay kit includes a printed Bio-Plex Pro Assay Quick Guide (bulletin
#10028208), which can be used to prepare and run a full 1 x 96-well assay
plate. Users can also download a copy at www.bio-rad.com/bio-plex.
Bead Regions
Bead regions for all analytes are listed in the Read Plate section.
Multiplexing Capabilities
Due to different sample dilution considerations, IgE assay should not be
multiplexed with other assays in the isotyping product line.
Multiplexing singleplex assays (IgA, IgM, and IgG total) is not recommended
due to lot differences of components. If assaying for IgA and IgM, then
consider running the 6-plex panel.
To avoid cross-reactivity, IgG total assay should not be multiplexed with any
of its subclasses (IgG1, IgG2, IgG3, and IgG4).
Detailed Instructions
The following pages provide detailed instructions for each step of the
assay procedure, including preparation, running the assay, and reading the
plate with Bio-Plex Manager™ and Luminex xPonent software.
7
1. Plan Plate Layout
Prior to running the assay, determine the total number of wells in the
experiment using the Plate Layout Template on page 33 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 your
chosen isotyping diluent or a diluent similar to your final sample type
or matrix. Note that Bio-Plex Manager automatically subtracts the
blank (B) MFI value from all other assay wells.
3.Controls, either user-specified or the quality controls supplied, are
assigned to wells in columns 3 and 4.
4. The remainder of the plate is available for samples.
5. Once the total number of wells is known, calculate the required volumes
of beads, detection antibody, and streptavidin-PE needed. Use Tables 6,
8, and 9, respectively, or the Calculation Worksheet on page 34.
Legend
S
Standard
B
Blank
X
Samples
C
Controls
Fig. 2. Suggested plate layout. For detailed instructions on plate
formatting in Bio-Plex Manager software, see section Read Plate.
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.
Note: While the instrument is warming up, bring the 10x wash buffer,
assay buffer, and isotyping diluent to room temperature. Keep other items
on ice until needed. Also, begin to thaw frozen samples.
Start up and calibrate the Bio-Plex system with Bio-Plex Manager™
software prior to setting up the assay. The calibration kit should be run
daily or before each use of the instrument to standardize the fluorescent
signal. For instructions on using other xMAP system software packages,
contact Bio-Rad Technical Support.
The validation kit should be run monthly to ensure optimal performance of
fluidics and optics systems. Refer to either the software manual or online
Help for directions on how to conduct validation.
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 Wash Buffer
1. Bring the 10x stock solution to room temperature.
2.If crystals exist, ensure that they are completely dissolved. Mix the
10x stock solution by inversion before preparing the 1x wash buffer.
3.To prepare 1x wash buffer, dilute 1 part of 10x stock solution with
9 parts of deionized water.
5. Prepare Standards and Controls
General Instructions
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n
It is essential to prepare standards and quality controls exactly as
described in this section. Incorrect preparation may lead to low signal or
variable measurements from plate to plate
The product data sheet provided lists the most concentrated point on the
standard curve (S1). Enter the values and units into Bio-Plex Manager™
software as instructed in section 9
Using the Quality Controls (optional)
One vial of quality controls is included. Its use is intended for monitoring
the day-to-day quality of assay results.
11
Selecting a Diluent for Standards and Controls
Refer to Table 4 for recommended diluents based on different sample types.
In order to meet the lot-specific control ranges provided on the product data
sheet, both the standards and controls should be reconstituted in Bio-Plex®
isotyping diluent. If reconstituting in a different diluent, users will need to
establish/validate their own control ranges or acceptance criteria.
Table 4. Summary of recommended diluents for samples.
Sample Type
Diluent for Standard and Controls* Add BSA
Serum and plasma
Culture media, with serum
Culture media, serum-free
Isotyping diluent
Culture media
Culture media
None
None
To 0.5% final
* If using diluents other than the isotyping diluent provided, then users must establish their
own control ranges.
Reconstitute Standards and Quality Controls
This procedure prepares enough standard to run each dilution in duplicate.
Note: The appearance of the lyophilized standards or controls may vary
from a white pellet to clear crystals. Regardless of appearance, the vials
have passed QC specifications and perform accordingly.
1.Gently tap the vial of lyophilized standards on a solid surface to
ensure that all material is at the bottom of the vial.
2.Reconstitute the vial of standards with 781 µl of the appropriate diluent.
Optional: at the same time, reconstitute the vial of controls with 250 µl
of the appropriate diluent as summarized in Table 4. Controls do not
require further dilution. If using diluents other than the isotyping
diluent provided, then users must establish their own control ranges.
3. Gently vortex the reconstituted standards and controls for 5 sec,
then incubate on ice for 30 min. It is important that reconstitution
of standards and controls is started and ended at the same time.
Be consistent with this incubation time to ensure optimal assay
performance and reproducibility.
4. During the incubation period, prepare the samples as instructed in the
Prepare Samples section.
12
Prepare the Standard Dilution Series
The following procedure produces an eight-point standard curve with a
fourfold dilution between each point. Pipet carefully using calibrated pipets
and use a new pipet tip for every volume transfer.
1.Label eight 1.5 ml microcentrifuge tubes S2 through S8 and Blank.
Alternatively, using Titertube® micro test tubes may prove to be more
convenient if a multichannel pipet will be used to load the plate.
2.Add 150 µl of the appropriate diluent to tubes S2–S8 and Blank.
(Figure 3).
3. V
ortex reconstituted standards at medium speed for 5 sec before
removing any volume. Transfer 50 µl to the S2 tube containing the
chosen standard diluent. Vortex for 5 sec.
4. Use a new pipet tip to transfer 50 µl from the S2 tube to the
S3 tube. Vortex for 5 sec.
5. Continue with 1:4 (fourfold) serial dilutions as shown in Figure 3.
6. Use reconstituted and diluted standards and controls immediately. Do
not freeze for future use.
Fig. 3. Preparing a fourfold dilution series with a single reconstituted standard.
50
Reconstituted
Standard
S1
50 505050 5050
150 150 150 150150 150150150
S2 S3S4S5S6S7S8
Blank
13
Transfer Volume, µl
Diluent, µl
6. Prepare Samples
These assays are designed to quantitate classes and subclasses of
immunoglobulins in serum, plasma, and cell culture media. For optimal
recovery and sensitivity, it is important to properly prepare samples.
n
Once thawed, keep samples on ice. Prepare dilutions just prior to the
start of the assay and equilibrate to room temperature before use
Prepare sample dilutions in 1.5 or 2 ml microcentrifuge tubes. If a
multichannel pipet will be used to load the plate, then aliquot the required
volumes into Titertube® micro test tubes.
n
Do not freeze diluted samples
n
Table 5. Summary of recommended sample diluents and dilution factors.
Sample Type
Diluent
Add BSA
Recommended Sample Dilution
Serum and plasma
Isotyping
None
diluent
1:40,000 for IgG1, IgG2, IgG3, IgG4,
IgA, IgM, IgG total
1:500 for IgE
Culture media, with serum Culture media None
User defined
Culture media, serum-free
Culture media To 0.5% final User defined
Sample Preparation
Serum and Plasma
EDTA or citrate is preferred as an anticoagulant. Heparin-treated plasma,
while compatible with Bio-Plex Pro™ assays, may absorb certain soluble
proteins of interest. Avoid using hemolyzed samples as this may lead to
false positive results.
1. Draw whole blood into collection tubes containing anticoagulant. Invert tubes several times to mix.
2.For serum, allow blood to clot at room temperature for 30 to 45 min.
For plasma, proceed directly to the centrifugation steps.
3. Perform centrifugation at 1,000 x g for 15 min at 4°C and transfer the
serum or plasma to a clean polypropylene tube.
14
4.Extremely lipemic samples may be filtered through a 0.2 µm filter to
prevent clogging.
5.Dilute samples with isotyping diluent following a 2-step dilution scheme
as noted below, vortexing for 5 sec between each dilution step.
1:40,000 dilution for IgG1, IgG2, IgG3, IgG4, IgA, IgM, and IgG total
Step 1. 5 µl of sample + 995 µl of diluent
Step 2. 5 µl of solution from first step + 995 µl of diluent
1:500 dilution for IgE
Step 1. 5 µl of sample + 120 µl of diluent
Step 2. 10 µl of solution from first step + 190 µl of diluent
6. Assay samples immediately or aliquot into single-use tubes and store at –70°C. Avoid repeated freeze-thaw cycles.
Cell Culture Supernatant
1. Collect supernatants and centrifuge at 1,000 x g for 15 min at 4°C.
For cell lines cultured in serum-free culture media, collect samples
and add BSA as a carrier protein to a final concentration of 0.5% to
stabilize protein analytes and to prevent adsorption to labware.
2. Transfer to a clean polypropylene tube. If cellular debris or precipitates are present, centrifuge again at 10,000 x g for 10 min at 4°C.
3. Reconstitute and dilute the isotyping standard in the same medium
or matrix in which cells are prepared. Be sure to include all medium
components (such as FBS) as appropriate. To minimize error due
to lot-to-lot variation of culture media, use the same lot of culture
medium that was used to prepare the cells.
4. Assay immediately or store samples in single-use aliquots at –70°C.
Avoid repeated freeze-thaw cycles.
15
7. Prepare Coupled Beads
1.Use Table 6 or the Calculation Worksheet on page 34 to calculate the
volume of coupled beads and assay buffer needed.
2. Add the required volume of Bio-Plex® assay buffer to a 15 ml
polypropylene tube.
3. Vortex the 20x stock of coupled beads at medium speed for 30 sec.
Carefully open the cap and pipet any liquid trapped in the cap back
into the tube. This is important to ensure maximum bead recovery.
Do not centrifuge the vial; doing so will cause the beads to pellet.
4.Dilute coupled beads to 1x by pipetting the required volume into the
15 ml tube. Vortex.
Each well of the assay requires 2.5 μl of the 20x stock adjusted to a
final volume of 50 μl in assay buffer.
5. Protect the beads from light with aluminum foil. Equilibrate to room
temperature prior to use.
Note: To minimize volume loss, use a 200–300 μl capacity pipet to remove
beads from the 20x stock tube. If necessary, perform the volume transfer in
two steps. Do not use a 1,000 μl capacity pipet and/or wide bore pipet tip.
Table 6. Preparing 1x coupled beads from 20x stock (includes 20% excess volume).
20x Beads, µl Assay Buffer, µl 96
# of Wells
288
5,472
5,760
48
144
2,736
2,880
16
Total Volume, µl
8. Run Assay
Considerations
n
n
n
n
Bring all buffers, diluents, diluted standards, diluted coupled beads, 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 at 850 ± 50 rpm. Cover
the plate with sealing tape and protect from light with aluminum foil
Table 7. Summary of wash steps and incubations. After each assay step, select the
appropriate Bio-Plex Pro™ wash station program or perform the appropriate manual wash step
as summarized below.
Bio-Plex Pro or Pro II Wash Station
Bio-Plex Pro II
Wash Station Handheld Magnet or
Vacuum Manifold
Assay Step
Magnetic Program
Vacuum Program
Manual Wash Steps
MAG x2
VAC x2
2 x 100 μl
Sample incubation
Detection Ab incubation
MAG x3
VAC x3
SA-PE incubation
3 x 100 μl
Add beads to plate
Considerations When Using a Vacuum Manifold
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 sheet of sealing
tape. Avoid pressing down over the wells to prevent leaking from the bottom
n
17
Add Coupled Beads, Samples, Standards, Blank,
and Controls
1. Cover unused wells of the assay plate with sealing tape.
2. Prewet the filter plate. Skip this step if using a flat bottom plate.
a) Prewet the wells with 100 µl assay buffer and remove the liquid
by vacuum filtration. Dry the bottom of the filter plate thoroughly
by blotting on a clean paper towel.
3. Vortex the diluted (1x) beads for 30 sec at medium speed. Pour into
a reagent reservoir and transfer 50 µl to each well of the assay plate.
TIP: A multichannel pipet is highly recommended for ease of use
and efficiency.
4.Wash the plate two times with 100 µl Bio-Plex® wash buffer
according to your wash method of choice.
5.Vortex the diluted samples, standards, blank, and controls for 5 sec.
Transfer 50 µl of each to the appropriate well of the assay plate,
changing the pipet tip after every volume transfer.
6. Cover with a new sheet of sealing tape and incubate in the dark for
1 hr at room temperature with shaking at 850 ± 50 rpm.
Prepare and Add Detection Antibodies
1. While the samples are incubating use Table 8 or the Calculation
Worksheet on page 34 to calculate the volume of detection
antibodies and Bio-Plex detection antibody diluent needed. Detection
antibodies should be prepared 10 min before use.
2. Add the required volume of Bio-Plex detection antibody diluent to a
15 ml polypropylene tube.
3. Vortex the 20x stock of detection antibodies for 15 sec at medium
speed, then perform a 30 sec spin to collect the entire volume at the
bottom of the tube.
18
4. Dilute detection antibodies to 1x by pipetting the required volume into
the 15 ml tube. Vortex.
Each well of the assay requires 1.25 μl of the 20x stock adjusted to a
final volume of 25 μl in detection antibody diluent.
Table 8. Preparing 1x detection antibodies from 20x stock (includes 25% excess volume).
# of Wells
20x Detection
Antibodies, µl Detection Antibody
Diluent, µl
96
150
2,850
3,000
48
75
1,425
1,500
Total Volume, µl
5.After incubating the beads, samples, standards, blank, and controls,
slowly remove and discard the sealing tape.
6. W
ash the plate three times with 100 µl wash buffer according to the
wash method of choice.
7. V
ortex the diluted (1x) detection antibodies gently for 5 sec. Pour into
a reagent reservoir and transfer 25 µl to each well of the assay plate
using a multichannel pipet.
8.Cover with a new sheet of sealing tape and incubate in the dark for
30 min at room temperature with shaking at 850 ± 50 rpm.
Prepare and Add Streptavidin-PE (SA-PE)
1.While detection antibodies are incubating, use Table 9 or the Calculation
Worksheet on page 34 to calculate the volume of SA-PE and assay
buffer needed. SA-PE should be prepared 10 min before use.
2. Add the required volume of assay buffer to a 15 ml polypropylene tube.
3.Vortex the 100x stock of SA-PE for 5 sec at medium speed. Perform
a 30 sec spin to collect the entire volume at the bottom of the vial.
19
4.Dilute SA-PE to 1x by pipetting the required volume into the 15 ml
tube. Vortex and protect from light until ready to use.
Each well of the assay requires 0.5 μl of the 100x stock adjusted to a
final volume of 50 μl in assay buffer.
Table 9. Preparing 1x SA-PE from 100x stock (includes 25% excess volume).
# of Wells
100x SA-PE, µl
Assay Buffer, µl
Total Volume, µl
96
60
5,940
6,000
48
30
2,970
3,000
5.After detection antibody incubation, slowly remove and discard
the sealing tape.
6.Wash the plate three times with 100 µl of wash buffer according
to the wash method of choice.
7. Vortex the diluted (1x) SA-PE at medium speed for 5 sec. Pour
into a reagent reservoir and transfer 50 µl to each well using a
multichannel pipet.
8.Cover with a new sheet of sealing tape and incubate in the dark for
10 min at room temperature with shaking at 850 ± 50 rpm.
9. After the streptavidin-PE incubation step, slowly remove, then
discard the sealing tape.
10. Wash the plate three times with 100 µl of wash buffer according
to the wash method of choice.
11.To resuspend beads for plate reading, add 125 µl assay buffer to
each well. Cover the plate with a new sheet of sealing tape. Shake
at room temperature at 850 ± 50 rpm for 30 sec and slowly remove
the sealing tape. Ensure that the plate cover has been removed
before placing the plate on the reader.
20
9. Read Plate
Bio-Plex Manager™ software is recommended for all Bio-Plex Pro™ assay
data acquisition and analysis. Instructions for Luminex xPONENT software
are also included. For instructions using other xMAP system software
packages, contact Bio-Rad Technical Support or your regional Bio-Rad
field applications specialist.
Prepare Protocol in Bio-Plex Manager Software
Version 6.0 and Higher
The protocol should be prepared in advance so that the plate is read as
soon as the experiment is complete.
A protocol file specifies the analytes in the assay, the plate wells to be
read, sample information, the values of standards and controls, and
instrument settings.
Bio-Plex Manager software versions 6.0 and higher contain protocols for
most Bio-Plex® assays. Choose from available protocols or create a new
protocol. To create a new protocol, select File, then New from the main
menu. Locate and follow the steps under Protocol Settings.
1. C
lick Describe Protocol in the Protocol Settings bar and enter
information about the assay (optional).
2. C
lick 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 list. If using Bio-Plex Manager version 5.0 or lower, select MagPlex from the assay dropdown list.
21
b.
Click the Add button. Enter the bead region number and name
for the first analyte. Click Add Continue to repeat for each
analyte in the assay.
For reference, bead regions are shown in Table 10.
Table 10. Bead regions for Bio-Plex Pro isotyping assays.
Analyte
Bead Region
lgG154
lgG221
lgG336
72
lgG4
Analyte
Bead Region
lgA
lgM
lgE
lgG Total
12
56
38
66
c. Click the Add button when the last analyte has been added and
click OK to save the new panel.
d. Highlight analytes from the Available list (left) and move to the
Selected list (right) using the Add button. To move all analytes at
once, simply click the Add All button.
e.
If some of the analytes need to be removed from the Selected
list, highlight them and select Remove. If desired, it is possible to
rename the panel by clicking on Rename Panel and entering a
new panel name.
3.Click Format Plate and format according to the plate template
created in section 1 (Plan Plate Layout). To modify the plate layout,
follow the steps below (see Figure 4).
a. Select the Plate Formatting tab.
b.Select the standards icon S and drag the cursor over all
the wells that contain standards. Repeat this process for
blanks B , controls C , and samples X . Note that
Bio-Plex Manager automatically subtracts the blank FI
value from all other assay wells.
22
Fig. 4. Plate formatting.
4. Click Enter Standards Info.
a.Enter the highest concentration and units of each analyte in the
top row (labeled S1) of the table. S1 concentration information is
listed in the product data sheet.
b. Enter a dilution factor of 4 and click Calculate. The concentrations
for each standard point will be populated for all analytes in the table.
c. Optional: enter the lot number of the vial of standards into the
Standard Lot box and click Save.
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.
23
b.
For the vial of quality controls supplied, format the appropriate
wells as controls and enter descriptions, but leave the
concentrations blank. Alternatively, the quality 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 11.
Table 11. Read the plate using the appropriate instrument settings.
Instrument
RP1 (PMT)
DD Gates
Bead Events
Bio-Plex 100, 200*
Low 5,000 (low), 25,000 (high) 50
Bio-Plex 3D*
Standard
Select MagPlex beads 50
Bio-Plex® MAGPIX™
N/A, use default instrument settings
* A similar Luminex-based system may be used.
a.Confirm that data acquisition is set to 50 beads per region.
b.In Bio-Plex Manager software prior to 6.1, go to Advanced
Settings and confirm that the bead map is set to 100 region, the
sample size is set to 50 µl, and the doublet discriminator (DD) gates
are set to 5,000 (Low) and 25,000 (High). In Bio-Plex Manager
software versions 4.0, 4.1, 4.1.1, and 5.0, check Override Gates
and set the DD gate values as indicated.
c.
Select Start, name and save the .rbx file, and begin data
acquisition. The Run Protocol pop-up screen will appear.
Click Eject/Retract to eject the plate carrier.
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.
24
2.Run Protocol — on the pop-up screen, select Load Plate and click
OK to start acquiring data.
3. Use the Wash Between Plates
command after every plate run
to reduce the possibility of clogging the instrument.
4.
If acquiring data from more than one plate, empty the waste bottle
and refill the sheath bottle after each plate (if HTF are not present).
Select Wash Between Plates and follow the instructions. Then repeat
the Prepare Protocol and Acquire Data instructions.
5. When data acquisition is complete, select Shut Down
follow the instructions.
and
Reacquire Data
It is possible to acquire data from a well or plate a second time using the
Rerun/Recovery mode located below Start in the Run Protocol step.
Any previous data will be overwritten.
1. Check the wells from which data will be reacquired.
2.Aspirate the buffer with the wash method of choice, but do not
perform wash step.
3. Add 100 µl of assay buffer to each well. Cover the plate with a new
sheet of sealing tape.
4. Repeat the Acquire Data steps to reacquire data. The data acquired
should be similar to those acquired initially; however, the acquisition
time will be extended because the wells have fewer beads.
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.
25
Note: Expected control ranges are provided for reference and should be
used as general guidelines. Actual results may vary for some operators.
If the controls do not fall within the expected ranges, please refer to the
troubleshooting section for possible causes and solutions.
Removing Outliers
Outliers are identified as standard data points that do not meet accuracy
or precision requirements and should be considered invalid when
performing curve fitting. As such, they should be removed to generate a
more realistic and accurate standard curve. This may result in an extended
assay working range and allow quantitation of samples that might
otherwise be considered out of range.
In Bio-Plex Manager software version 6.0 and higher, outliers can be
automatically removed by selecting the Optimize button in the Standard
Curve window. In Bio-Plex Manager software 6.0 and earlier versions,
outliers can be manually selected in the Report Table. Visit online Help to
learn more about the standard curve optimizer feature and how outliers
are determined.
Previous Versions of Bio-Plex Manager Software
For instructions on using previous versions of Bio-Plex Manager software,
please contact Bio-Rad Technical Support.
In Depth Data Analysis
To better understand your data’s biological significance, consider using
Bio-Plex Data Pro™ software, which makes data management and
comparison easier. This software offers various graphing options and
automatic calculations with simple, conservative statistical calculations.
Contact your local Bio-Rad representative for more information.
26
Luminex xPONENT Software
Luminex xPONENT software may be used to analyze Bio-Plex assays.
Although guidelines are provided here, consult the xPONENT software
manual for more details. Perform a system initialization with Luminex’s
calibration and performance verification kit, as directed by Luminex.
Select Batches to set up the protocol and follow the information
under Settings.
Note: The instrument settings described below apply to Luminex 100/200
and FLEXMAP or Bio-Plex 3D instruments. For the Bio-Plex MAGPIX
reader, use the default instrument settings.
1. Select MagPlex as the bead type for magnetic beads. This
automatically sets the DD gates.
2. Volume = 50 µl.
3.Refer to Table 11 to select the appropriate PMT setting for your
instrument.
4. Plate name: 96-well plate.
5. Analysis type: Quantitative, 5PL Curve Fit.
6. Number of standards: 8.
Select Analytes to set up the panel.
1. Enter “ng/ml” in the Units field.
2. Enter 50 in the Count field.
3. Select the bead region and enter the analyte name.
4. Click Apply all for Units and Count.
Select Stds and Ctrls.
1. Enter standard concentrations, lot number, dilution factor, and other
information as applicable.
After the assay is complete, select Results, then select Saved Batches.
27
Troubleshooting Guide
This troubleshooting guide addresses problems that may be encountered
with Bio-Plex Pro™ assays. If you experience any of the problems listed
below, review the possible causes and solutions provided. Poor assay
performance may also be due to the Bio-Plex® suspension array reader.
To eliminate this possibility, use the validation kit to assist in determining if
the array reader is functioning properly.
Possible Causes
High Inter-Assay CV Standards and controls were not
reconstituted consistently between
assays
Possible Solutions
Reconstituted standards, controls,
and diluted samples were not
stored properly
Reconstituted standards and diluted
samples should be prepared on ice
as instructed. Prior to plating, the
reconstituted standards and diluted
samples should be equilibrated to
room temperature.
Bottom of filter plate not dry
Dry the bottom of the filter plate with
absorbent paper towel (preferably
lint-free) to prevent cross-well
contamination.
Incubate the reconstituted
standards for 30 min on ice. Always
be consistent with the incubation
time and temperature.
28
Possible Causes
Possible Solutions
High Intra-Assay CV
Improper pipetting technique
Reagents and assay components
not equilibrated to room
temperature prior to pipetting
Contamination with wash buffer
during wash steps
Pipet carefully when adding
standards, controls, samples,
detection antibodies, and
streptavidin-PE, especially when
using a multichannel pipet. Use a
calibrated pipet. Change pipet tip
after every volume transfer.
All reagents and assay components
should be equilibrated to room
temperature prior to pipetting.
During the wash steps, be careful
not to splash wash buffer from one
well to another. Be sure that the
wells are filtered completely and that
no residual volume remains. Ensure
that the microplate shaker setting is
not too high. Reduce the microplate
shaker speed to minimize splashing
Slow pipetting of samples and
reagents across the plate
Sample pipetting across the entire
plate should take less than 4 min.
Reagent pipetting across the entire
plate should take less than 1 min.
29
Possible Causes
Low Bead Count
Miscalculation of bead dilution
Possible Solutions
Beads clumped in multiplex
bead stock tube
Vortex for 30 sec at medium speed
before aliquoting beads.
Vacuum on for too long when
aspirating buffer from wells
Assay plate not shaken enough
during incubation steps and prior
to reading
Reader is clogged
Do not apply vacuum to the filter
plate for longer than 10 sec after the
buffer is completely drained from
each well.
Incorrect needle height of the
reader
Adjust the needle height to coincide
with the plate type provided in the kit.
Check your calculations and be
careful to add the correct volumes.
Shake the plate at 850 ± 50 rpm
during incubation steps and for
30 sec immediately before reading
the plate.
Refer to the troubleshooting guide
in the Bio-Plex® system hardware
instruction manual (bulletin
#10005042).
Low Signal or Poor Sensitivity
Standards reconstituted incorrectly Follow the instructions carefully.
Detection antibody or Check your calculations and be
streptavidin-PE diluted incorrectly
careful to add the correct volumes.
30
Possible Causes
Possible Solutions
Incorrect buffer was used
(for example, assay buffer used
to dilute standards)
Use isotyping diluent to dilute
standards.
High Background Signal
Accidentally spiked blank wells
Detection antibodies or
streptavidin-PE incubated too long
Poor Recovery
Expired Bio-Plex reagents
were used
Incorrect amounts of components
were added
Do not add any antigens to the
blank wells.
Follow the procedure incubation
time precisely.
Check that reagents have not expired.
Use new or nonexpired components.
Check your calculations and be
careful to add the correct volumes.
Microplate shaker set to an
incorrect speed
Check the microplate shaker speed
and use the recommended setting.
Setting the speed too high may
cause splashing and contamination.
Use the recommended plate shaker.
High end saturation of the
standard curve
Make sure that correct shaker speed
and incubation times are used.
Remove S1 for data analysis if needed.
Quality controls do not fall within
expected ranges.
Make sure that the vial of controls
is reconstituted at the same time as
standards and in the same diluent.
Incubate for precisely 30 min.
31
Possible Causes
Possible Solutions
Poor Recovery
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.
Impact of Sample Matrix
Negative MFI values in
samples or standards
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.
32
Plate Layout Template
33
Calculation Worksheet
If using either a premixed panel or one singleplex assay, follow these directions.
Plan the plate layout and enter the number of wells to be used in the assay:_______
1
1. Determine the volume of 1x coupled beads needed.
a. Each well requires 50 µl of coupled beads (1x): _______ x 50 µl = _______ µl
12
b. Include 20% excess to ensure enough volume: _______ µl x 0.20 = _______ µl
23
c. Total volume of 1x coupled beads: _______ µl + _______ µl = _______ µl
234
d. Volume of 20x coupled beads required: _______ µl/20 = _______ µl
45
e. Volume of assay buffer required: _______ µl – _______ µl = _______ µl
45 6
2. Determine the volume of 1x detection antibody needed.
a. Each well requires 25 µl detection antibodies (1x): ______ x 25 µl = _______ µl
17
b. Include 25% excess to ensure enough volume: _______ µl x 0.25 = _______ µl
78
c. Total volume of 1x detection antibodies: _______ µl + _______ µl = _______ µl
78 9
d. Volume of 20x detection antibodies required: _______ µl/20 = _______ µl
910
e. Volume of detection antibody diluent required: _____ µl – _____ µl = _____ µl
9 1011
3. Determine the volume of 1x streptavidin-PE needed.
a. Each well requires 50 µl streptavidin-PE (1x): _______ x 50 µl = _______ µl
110
b. Include 25% excess to ensure enough volume: _______ µl x 0.25 = _______ µl
1011
c. Total volume of 1x streptavidin-PE: ______ µl + ______ µl = ______ µl
101112
d. Volume of 100x streptavidin-PE required: _______ µl/100 = _______ µl
1213
e. Volume of assay buffer required: _______ µl – _______ µl = _______ µl
1213 14
34
Safety Considerations
Eye protection and gloves are recommended when using these products.
Consult the MSDS for additional information. The Bio-Plex Pro™ assays
contain components of animal origin. This material should be handled as
if capable of transmitting infectious agents. Use universal precautions.
These components should be handled at Biosafety Level 2 containment
as defined by U.S. government publication, Biosafety in Microbiological
and Biomedical Laboratories (Centers for Disease Control, 1999).
Legal Notices
Acrodisc and Supor are trademarks of Pall Corporation. MagPlex, xMAP,
xPONENT, and Luminex are trademarks of Luminex Corporation.
The Bio-Plex® suspension array system includes fluorescently labeled
microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc.
by the Luminex Corporation.
35
Ordering Information
Detailed ordering information can be found at www.bio-rad.com/bio-plex.
Catalog #
Premixed All-In-One Multiplex Kit
Premixed multiplex kit includes coupled magnetic beads, detection antibodies, standards, 1-level quality controls,
detection antibody diluent, isotyping diluent (for use with samples, standards, and controls), assay buffer, wash
buffer, streptavidin-PE, 96-well flat bottom plate, sealing tape, assay quick guide, and product data sheet
171-A3100M Bio-Plex Pro Human Isotyping Panel, 6-plex, 1 x 96-well, for the detection of IgG1,
IgG2, IgG3, IgG4, IgA, IgM
Catalog #
All-In-One Singleplex Kits
Singleplex kits are specific for each assay indicated below and include coupled magnetic beads, detection
antibodies, standards, 1-level quality controls, detection antibody diluent, isotyping diluent (for use with samples,
standards, and controls), assay buffer, wash buffer, streptavidin-PE, 96-well flat bottom plate, sealing tape, assay
quick guide, and product data sheet
171-A3101M
171-A3102M
171-A3104M
171-A3103M
Bio-Plex Pro Human IgA Isotyping Assay
Bio-Plex Pro Human IgE Isotyping Assay
Bio-Plex Pro Human IgM Isotyping Assay
Bio-Plex Pro Human IgG Total Isotyping Assay
Catalog #
Wash Stations and Accessoriess
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
171-025001
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
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
Catalog #
Software
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) Standard Edition, for instrument data evaluation
and optimization
36
Bio-Rad
Laboratories, Inc.
Life Science
Group
Web site www.bio-rad.com USA 800 424 6723 Australia 61 2 9914 2800
Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 11 5044 5699
Canada 905 364 3435 China 86 21 6169 8500
Czech Republic 420 241 430 532 Denmark 44 52 10 00
Finland 09 804 22 00 France 01 47 95 69 65 Germany 089 31 884 0
Greece 30 210 9532 220 Hong Kong 852 2789 3300
Hungary 36 1 459 6100 India 91 124 4029300 Israel 03 963 6050
Italy 39 02 216091 Japan 03 6361 7000 Korea 82 2 3473 4460
Mexico 52 555 488 7670 The Netherlands 0318 540666
New Zealand 64 9 415 2280 Norway 23 38 41 30
Poland 48 22 331 99 99 Portugal 351 21 472 7700
Russia 7 495 721 14 04 Singapore 65 6415 3188
South Africa 27 861 246 723 Spain 34 91 590 5200
Sweden 08 555 12700 Switzerland 026 674 55 05
Taiwan 886 2 2578 7189 Thailand 800 88 22 88
United Kingdom 020 8328 2000
10028370 Rev B
Sig 1212
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