10011597

10011597
Bio-Plex Manager™ software
recommended
TM
Bio-Plex Pro Assays
Acute Phase
Instruction Manual
For technical support, contact your local Bio-Rad office or
in the US, call 1-800-4BIORAD (1-800-424-6723).
For research use only. Not for diagnostic procedures.
Table of Contents
Section 1
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Section 2
Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Section 3
Bio-Plex® Acute Phase Reagent Kit Product Description .4
Section 4
Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Section 5
Recommended or Optional Materials . . . . . . . . . . . . . 6
Section 6
Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Section 7
Standard Preparation . . . . . . . . . . . . . . . . . . . . . . . . 10
Section 8
Control Preparation . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section 9
Assay Instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Section 10
Data Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Section 11
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . 30
Section 12
Safety Considerations . . . . . . . . . . . . . . . . . . . . . . . .34
Section 1
Introduction
Acute phase proteins are a class of proteins that have altered levels in
response to inflammation and are thus relevant biomarkers in many
disease processes. Typically, initial release of cytokines at the site of
damage, most notably IL-1, IL-6, IL-8 and TNF-움, triggers the liver to
release a number of acute phase proteins into the bloodstream. In turn,
the increased levels of these proteins stimulate the release of more
cytokines, presumably to initiate processes to prevent further tissue
damage. Positive acute phase proteins—those that increase in response
to tissue damage—are associated with infection, physical trauma, cardiac
injury, diabetes, cancer, and other diseases.
Bio-Plex Pro™ assays are bead-based multiplex assays that allow the
measurement of nine positive acute phase biomarkers in diverse matrices,
including serum, plasma, and tissue culture supernatant. The biomarkers
selected are circulating proteins that are known to be present in elevated
levels during the acute phase response. The multiplexing feature makes it
possible to quantify the levels of multiple target proteins simultaneously in
a single well of a 96-well microplate in just 3 hours with as little as 5 µl of
sample.
As one of the most recent additions to the Bio-Plex family of products,
Bio-Plex Pro assays incorporate magnetic beads into their design. The
magnetic beads allow the use of an assay protocol similar to nonmagnetic
Bio-Plex assays, with the option of using magnetic separation during wash
steps instead of vacuum filtration. The magnetic washing option also
enables automated assay preparation.
Bio-Plex Manager™ software is recommended for Bio-Plex Pro acute phase
assays. Instructions for Luminex xPONENT software are also provided. For
instructions using other xMAP system software packages, contact Bio-Rad
Technical Support or your Bio-Rad Field Applications Scientist.
For a current listing of Bio-Plex Pro acute phase assays, visit us on the
Web at www.bio-rad.com/bio-plex/
1
Section 2
Principle
Technology
The Bio-Plex® suspension array system is built around three core
technologies. The first is a novel technology that uses up to 100 unique
fluorescently dyed beads (xMAP technology) that permit the simultaneous
detection of up to 100 different types of molecules in a single well of a
96-well microplate. The second is a flow cytometer with two lasers and
associated optics to measure the different molecules bound to the
surface of the beads. The third is a high-speed digital signal processor
that efficiently manages the fluorescent output.
Assay Format
The principle of these 96-well plate-formatted, bead-based assays is similar
to a capture sandwich immunoassay. An antibody directed against the
desired target protein is covalently coupled to internally dyed beads. The
coupled beads are allowed to react with a sample containing the target
protein. After a series of washes to remove unbound protein, a biotinylated
detection antibody specific for a different epitope is added to the reaction.
The result is the formation of a sandwich of antibodies around the target
protein. Streptavidin-phycoerythrin (streptavidin-PE) is then added to bind to
the biotinylated detection antibodies on the bead surface for flourescencebased detection.
Data Acquisition and Analysis
Data from the reaction are then acquired using the Bio-Plex suspension
array system, dual-laser, flow-based microplate reader system. The
contents of the well are drawn up into the reader. The lasers and
associated optics detect the internal fluorescence of the individual dyed
beads as well as the fluorescent signal on the bead surface that
corresponds to the amount of detected target protein. Intensity of
fluorescence detected on the beads indicates the relative quantity of
targeted molecules. A high-speed digital processor efficiently manages
the data output, which is further analyzed and presented as fluorescence
intensity on Bio-Plex Manager™ software.
2
Assay Workflow
Prewet wells
Add beads
Wash
Add standards, controls,
and samples, 60 min
Wash
Add detection antibody, 30 min
Wash
Add streptavidin-PE, 10 min
Wash
Resuspend, acquire data
3
Section 3
Bio-Plex® Acute Phase Reagent Kit
Product Description
Bio-Plex Pro™ acute phase assays require the use of the Bio-Plex acute phase
reagent kit.
Catalog #
171-304050
Catalog #
171-304051
1 x 96-Well
Format
10 x 96-Well
Format
Bio-Plex assay buffer
Store at 4ºC. Do not freeze.
1 x 75 ml
1 x 750 ml
Bio-Plex wash buffer
Store at 4ºC. Do not freeze.
1 x 150 ml
2 x 750 ml
Bio-Plex detection
antibody diluent
Store at 4ºC. Do not freeze.
1 x 15 ml
1 x 150 ml
Streptavidin-PE (100x)
Store at 4ºC. Do not freeze.
1 vial
1 vial
1 plate
10 plates
1 pack of 4
10 packs of 4 (40)
1
1
Component of the
Bio-Plex Acute Phase
Reagent Kit
Sterile filter plate (96-well)
with cover and tray
Sealing tape
Acute Phase instruction
manual
Storage and Stability
Kit components should be stored at 4ºC and never frozen. Coupled
magnetic beads and streptavidin-PE should be stored in the dark. All
components are guaranteed for up to 6 months from the date of
purchase when stored as specified in this manual.
4
Section 4
Required Materials
The Bio-Plex Pro™ acute phase assay panel is divided into two separate
multiplex assay kits based on the sample dilution required for optimal
performance.
•
Each assay requires one Bio Plex® acute phase reagent kit.
•
The Bio-Plex acute phase diluent kit (see next section) is
recommended for serum and plasma samples. One diluent
kit contains sufficient volumes for performing a 5-plex assay
and a 4-plex assay or two 5-plex assays or two 4-plex
assays, as long as dilutions are performed as instructed.
The Bio Plex Pro acute phase assay kits contain the following components:
Multiplex assays
• Antibody-conjugated beads (25x concentration)
•
Detection antibody (10x concentration)
•
Acute phase standard (2 vials/lyophilized)
•
Acute phase control (2 vials/lyophilized)
Please visit the Bio-Plex web site at www.bio-rad.com/bio-plex/ for our
current list of assays and panels.
Bio-Plex Manager™ software version 3.0 and Luminex IS Software Users
Contact Bio-Rad Technical Support or your Bio-Rad Field Applications
Scientist. Additional supplies are required to run the acute phase assays
using these systems.
5
Section 5
Recommended or Optional Materials
Item
Catalog #
®
Bio-Plex Acute Phase Diluent Kit
Serum-based diluent
Serum-free diluent
Bio-Plex Pro™ Human Acute Phase
Standards
Optional additional standards sold
separately
Item
(300 ml)
171-D40002
(2 vials, lyophilized)
171-D00006
(50 vials, lyophilized)
Ordering Information
Bio-Plex 200 System with HTF
(or Luminex System)
Bio-Rad catalog #171-000205
Bio-Plex Validation Kit 4.0
Bio-Rad catalog #171-203001
Bio-Plex Calibration Kit
Bio-Rad catalog #171-203060
Microtiter Plate Shaker
IKA-Schuttler MTS-4 shaker for 4
microplates or Lab-Line Model 4625
plate shaker (or equivalent, capable of
300-1,100 rpm)
Filter Plate Vacuum Apparatus
Aurum™ Vacuum Manifold
IMPORTANT: The use of filter plate
manifolds other than the one specified
may result in diminished assay
performance; see section 8 for
instructions specific to this assay
Vortexer
VWR mini-vortexer
Scientific Instruments Vortex-Genie 2 mixer
6
171-305050 (1x96)
(10 ml)
IKA catalog #3208000
VWR catalog #57019-600
Bio-Rad catalog #732-6470
VWR catalog #58816-121
VWR catalog #58815-234
Item (continued)
Reagent Reservoir
Corning, Inc. Costar 50 ml reagent
reservoir 4870
1.2 ml Tubes in 96 Rack
For performing sample dilutions
in 96-well format
Ordering Information
Bio-Rad catalog #224-4872
VWR catalog #82006-698
(tubes in rack)
VWR catalog #83009-678
(refill tubes)
Other Materials
Pipets and pipet tips, sterile distilled
water, aluminum foil, absorbent paper
towels, 1.5 ml microcentrifuge tubes,
15 ml culture tubes
7
Section 6
Sample Preparation
This section provides instructions for preparing samples derived from
serum, plasma, and culture supernatant. For sample preparations not
mentioned here, consult the publications listed in Bio-Rad bulletin 5297,
available for download at www.discover.bio-rad.com
Serum and Plasma Samples
Collect and process the serum or plasma samples and assay immediately
or freeze at –20ºC. Avoid repeated freeze-thaw cycles. EDTA is the
recommended anticoagulant for preparing plasma samples. Extremely
lipemic samples may be filtered with a 0.22 µm filter to prevent clogging.
Hemolyzed samples are not suitable for Bio-Plex Pro™ acute phase
assays.
1.
After collecting blood samples, immediately add appropriate
inhibitors according to manufacturer’s instructions if necessary, invert
tube several times to mix.
2.
Prior to assay setup, centrifuge the samples at 13,200 rpm for 10 min
at 4ºC to clear the samples of precipitate. Alternatively, carefully filter
the samples with a 0.22 µm filter to prevent instrument clogging.
Keep samples on ice until ready for use.
3.
Refer to the steps and the table below for sample dilution instructions
for preparing the 5-plex assay, the 4-plex assay, or for preparing
samples for the 4-plex and 5-plex assays at the same time.
5-plex assay: Dilute 5 µl of sample in 495 µl of serum free
diluent. Mix by vortexing gently or pipetting. This results in a
final 1:100 sample dilution.
4-plex assay: First dilute 5 µl of sample in 495 µl of serum-free
diluent to create a 1:100 dilution. Mix gently. Next, dilute 5 µl of
this mixture into an additional 495 µl of serum-free diluent. Mix
gently. This results in a final dilution of 1:10,000.
8
5-plex and 4-plex assays For the 5-plex assay, dilute 5 µl of
sample in 495 µl of serum-free diluent. Mix by vortexing gently
or pipetting. This results in a final 1:100 sample dilution. For the
4-plex assay, dilute 5 µl of the 1:100 mixture into an additional
495 µl of serum-free diluent. Mix gently. This results in a final
sample dilution of 1:10,000.
Panel Step Dilution
1 100-fold
5-plex
1 100-fold
4-plex
2 10,000-fold
Sample Volume
Serum-free
Diluent Volume
Total
Volume
5 μl sample
5 μl sample
5 μl of 1:100
495 μl
495 μl
495 μl
500μl
500μl
500μl
NOTE: Additional dilutions may be required in order for
samples with higher target concentrations to be within
assay range.
Culture Supernatant Samples
1. Collect and process the culture supernatant samples and assay
immediately or freeze at –20ºC. Avoid repeated freeze-thaw cycles.
2.
If required, dilute the culture supernatant with culture medium.
Serum-free culture medium should contain carrier protein (such as
BSA) at a concentration of at least 0.5%. Keep the samples on ice until
ready for use.
9
Section 7
Standard Preparation
Two vials of Iyophilized acute phase standards are provided in each
multiplex Bio-Plex Pro™ acute phase assay kit and one vial is provided in
each singleplex assay kit. Only one vial is required per 96-well plate. The
product insert provided with the assay lists the concentration of the most
concentrated dilution point of the standard curve. This procedure will
prepare enough standard to run each dilution in duplicate.
Reconstitute Standards
1. Reconstitute one 5-plex standard for every 5-plex assay and one 4plex standard for every 4-plex assay. Gently tap the glass vial
containing the lyophilized acute phase standards on a solid surface
to ensure that the pellet is on the bottom.
2.
Refer to the following steps for preparation of diluents for the 5-plex
and 4-plex standards and controls. Use the serum-based diluent and
serum-free diluent provided in the Bio-Plex® acute phase diluent kit
(catalog# 171-305050) for serum and plasma samples. For tissue
culture samples, use tissue culture medium. Do not use assay buffer
to dilute standards and controls.
5-plex standard/control diluent: Dilute 1 ml of serum-based
diluent in 24 ml of serum-free diluent. Vortex 10 sec. Label this
mixture "5-plex standard/control diluent".
4-plex standard/control diluent: The serum-free diluent is
used directly for diluting 4-plex standards and controls. Using a
sterile pipette, transfer 25 ml into a clean container labeled "4plex standard/control diluent".
3.
10
Reconstitute 1 standard vial per assay with 0.5 ml of the appropriate
diluent as described in Step 2. The mixtures are identified as S0 in
the Standard Dilution Series diagrams on page 11.
4.
Gently vortex 1-3 sec and incubate on ice for 60 min. For optimal
assay performance, be consistent with the incubation time.
5.
Save the standard/control diluents for preparation of the controls
(Section 8).
Prepare Standard Dilution Series
The standard concentrations specified for the 8-point standard dilution
set have been selected for optimized curve fitting using the 5-parameter
logistic (5PL) or 4-parameter logistic (4PL) regression in Bio-Plex Manager™
software. Results generated using dilution points other than those listed
in this manual have not been optimized.
1.
Label a set of 1.5 ml Eppendorf tubes as shown in the Standard
Dilution Series diagram on the next page.
2.
Pipet the appropriate volume of standard diluent into the tubes (see
diagram below).
3.
Refer to the steps and diagrams below for preparation of the
standard dilution series for the 5-plex and 4-plex assays.
5-plex standard dilution series: Add 200 µl of reconstituted 5-plex
standard to the first 1.5 ml tube containing 200 µl 5-plex standard
diluent. Vortex gently. This mixture is identified as S1 in the diagram
below and in the standard and control values product insert
provided with the assay. Continue making serial dilutions of the
standards as shown in the diagram. Before making each dilution,
vortex gently and change the pipet tip.
5-plex Standard Dilution Series
11
4-plex standard dilution series: Add 50 µl of reconstituted 4-plex
standard to the first 1.5 ml tube containing 350 µl 4-plex standard
diluent. Vortex gently. This mixture is identified as S1 in the diagram
below and in the standard and control values product insert provided
with the assay. Continue making serial dilutions of the standards as
shown in the diagram. Before making each dilution, vortex gently and
change the pipet tip.
4-plex Standard Dilution Series
Note: The addition of duplicate 0 pg/ml blanks is strongly
recommended. Use 50 µl of the appropriate standard diluent as the
blank sample. the 0 pg/ml points should be formatted as blanks, not
as points in the curve, when using Bio-Plex Manager software.
Formatting the 0 pg/ml wells as blanks results in an automatic
subtraction of the background Median Flourescence Intensity (MFI)
values from all readings in a run. This might result in negative MFI
values in some wells. If this is undesirable, the 0 pg/ml wells can be
formatted as background controls. The 0 pg/ml background control
wells are also useful for troubleshooting and determining the limit of
detection (LOD).
5.
12
Keep the standards on ice until ready for use. Standards should be
used immediately and should not be frozen for future use.
Section 8
Control Preparation
Two vials of lyophilized acute phase controls are provided in each
multiplex Bio-Plex Pro™ acute phase assay kit. This section provides
instructions on how to reconstitute the Iyophilized control. The product
insert provided with the assay lists the concentration ranges of the
prepared controls.
Prepare Acute Phase Controls
To ensure optimal assay performance, the acute phase controls should be
prepared in a manner consistent with that used to prepare the acute phase
standards.
1.
Gently tap the glass vial containing the lyophilized acute phase
control on a solid surface to ensure the pellet is at the bottom.
2.
Refer to the steps below for reconstituting the 5-plex and 4-plex
controls. Use the standard/control diluent prepared in the previous
section (Section 7, step 2). Do not use assay buffer to reconstitute
the controls.
5-plex control: Reconstitute one vial of lyophilized control with
1 ml of 5-plex standard/control diluent. This solution will be used
directly in the assay; no additional dilutions are required.
4-plex control: Reconstitute one vial of lyophilized control with
2 ml of 4-plex standard/control diluent. This solution will be used
directly in the assay; no additional dilutions are required.
3.
Gently vortex 1-3 sec and incubate on ice for 60 min. For optimal
assay performance, be consistent with the incubation time.
4.
Expected concentration ranges of the prepared controls are provided
in the standard and control values product insert.
13
Section 9
Assay Instructions
The following instructions apply to both the 5-plex and 4-plex assays. All
of the necessary components are provided premixed for ease of use.
Plan Experiment
1. Assign which wells of a 96-well plate will be used for each standard,
control, and sample (see the example plate layout below).
2.
Determine the total number of wells that will be used in the assay.
Include a 25% excess (or add 2 wells for every 8 wells used) to
ensure that enough diluted coupled beads, detection antibodies,
and streptavidin-PE are prepared.
Example Plate
14
Prepare Coupled Magnetic Beads
Protect the beads from light by covering the tubes with aluminum foil.
Keep all tubes on ice until ready to use.
1.
Vortex the coupled beads (25x) at medium speed for 30 sec.
2.
Prepare a sufficient volume of coupled beads (1x) using assay
buffer. Each well requires 2 µl of coupled beads (25x) adjusted to a
final volume of 50 µl with assay buffer (refer to the example below).
Example Bead Calculations
# of Wells
25x Beads (µl)
Assay Buffer (µl)
Total Volume (µl)
96
240
5,760
6,000
48
120
2,880
3,000
32
80
1,920
2,000
24
60
1,440
1,500
Calibrate Vacuum Apparatus
The vacuum apparatus must be calibrated at the beginning of the assay
to ensure an optimal bead yield. For more detailed instructions, refer to
the Bio-Plex® Suspension Array System hardware instruction manual.
1.
Prewet all the wells of a 96-well filter plate with 100 µl of assay buffer.
2.
Place the filter plate on the vacuum apparatus and turn on the
vacuum to the maximum level.
3.
Press on the filter plate and note the time required to remove the
buffer from the wells by vacuum filtration. The evacuation time
should be 2–5 sec.
If the evacuation time is <2 sec, the pressure is too high. Open the
vacuum control valve slightly and repeat steps 1–3.
If the evacuation time is >5 sec, the pressure is too low. Close the
vacuum control valve slightly and repeat steps 1–3.
15
Assay Procedure
Bring all buffers to room temperature. Avoid bubbles when pipetting.
The following terms are repeated throughout the assay procedure. Refer
to these detailed instructions when wash, incubate, and vacuum-filter are
shown in bold.
Detailed Directions
Add 100µL of wash buffer to each well. Place the filter plate on a calibrated vacuum apparatus and
remove the buffer by vacuum filtration. Blot the bottom of the filter plate with a clean paper towel.
Repeat as specified.
Tip: Thoroughly blot the bottom of the filter plate with a clean paper towel to prevent crosscontamination and plate leakage.
Gently cover the filter plate with a new sheet of sealing tape. Place the filter plate on a microplate
shaker and then cover with aluminum foil. Shake the filter plate at room temperature at 1,100 rpm
for 30 sec, then at 300 rpm for the specified incubation time.
Tip: Apply sealing tape gently on the filter plate (e.g. press down only on edges) to prevent positive
pressure inside the wells that could lead to plate leaking during shaking. To avoid splashing of
samples that may lead to cross-well contamination, slowly ramp up the shaker to the maximum
speed before incubation. Cover plate with aluminum foil to prevent photobleaching.
Place the filter plate on a calibrated vacuum apparatus and remove the buffer by vacuum filtration.
Blot the bottom of the filter plate with a clean paper towel.
Tip: Visually examine each well to ensure that buffers are completely drained from each well.
1.
Equilibrate the diluted standards, samples, and controls at room
temperature for 20 min prior to use.
2.
Prewet and block the desired number of wells in a 96-well filter plate
with 100 µl of assay buffer and vacuum-filter. If fewer than 96
wells are required, mark the plate to identify the unused wells for
later use and cover the unused wells with sealing tape.
3.
Vortex the coupled magnetic beads (1x) for 15–20 sec at medium
speed. Add 50 µl to each well and immediately vacuum-filter.
4.
Wash twice.
5.
Gently vortex the diluted standards, controls, and samples for 1–3 sec.
Add 50 µl of standard, control, or sample to each well, changing the
pipet tip after every volume transfer. Incubate for 1 hr.
16
6.
While the samples are incubating, perform a 30 sec quick-spin
centrifugation of the detection antibody (10x for multiplex assays,
100x for singleplex assays) prior to pipetting to collect the entire
required volume at the bottom of the vial.
7.
Prepare a sufficient volume of detection antibodies (1x) using
detection antibody diluent. Refer to the example tables below.
NOTE: The detection antibody stock concentration is 10x in the
multiplex assays and 100x in the singleplex assays. Always check
the detection antibody concentration on the vial label before diluting.
Example Detection Antibody Calculations (10x stock)
# of Wells
10x Detection
Antibody (µl)
Detection Antibody
Diluent (µl)
Total Volume (µl)
96
300
2,700
3,000
48
150
1,350
1,500
32
100
900
1,000
24
75
675
750
Example Detection Antibody Calculations (100x stock)
# of Wells
100x Detection
Antibody (µl)
Detection Antibody
Diluent (µl)
Total Volume (µl)
96
30
2,970
3,000
48
15
1,485
1,500
32
10
990
1,000
24
7.5
742.5
750
8.
After incubating the samples, slowly remove and discard the sealing
tape, then vacuum-filter.
9.
Wash 3 times.
10. Vortex the detection antibodies gently and add 25 µl to each well.
Incubate for 30 min.
17
11. While the detection antibodies are incubating, perform a 30 sec
quick-spin centrifugation of the streptavidin-PE (100x) prior to
pipetting to collect the entire volume at the bottom of the vial.
12. Prepare a sufficient volume of streptavidin-PE (1x) using assay buffer.
Each well requires 0.5 µl of streptavidin-PE (100x) adjusted to a final
volume of 50 µl with assay buffer (refer to the example below).
Example Streptavidin-PE Calculations
100x
Assay Buffer (µl)
Streptavidin-PE
# of Wells
(µl)
Total Volume (µl)
96
60
5,940
6,000
48
30
2,970
3,000
32
20
1,980
2,000
24
15
1,485
1,500
13. After the detection antibody incubation, slowly remove and discard
the sealing tape, then vacuum-filter.
14. Wash 3 times.
15. Vortex the streptavidin-PE (1x) vigorously and add 50 µl to each well.
Incubate for 10 min.
16. After the streptavidin-PE incubation, slowly remove and discard the
sealing tape, then vacuum-filter.
17. Wash 3 times.
18. Add 125 µl of assay buffer to each well. Cover the filter plate with a
new sheet of sealing tape. Shake the filter plate at room temperature
at 900 rpm for 30 sec and remove the sealing tape before
reading the plate.
18
Section 10
Data Acquisition
Bio-Plex Manager™ software is recommended for Bio-Plex Pro™ acute
phase assays. Refer to the details in the appropriate section below for
your xMAP system software package. Instructions for Luminex xPONENT
software are also provided.
For instructions using other xMAP system software packages, contact
Bio-Rad Technical Support or your Bio-Rad Field Applications Scientist.
NOTE: Bio-Plex Pro acute phase assays are analyzed using the low
RP1 target value (low PMT). Be sure to follow the instructions for your
software version on how to calibrate and analyze data.
NOTE: To minimize protocol setup, lot-specific Bio-Plex Pro acute
phase assay protocols for Bio-Plex Manager version 4.0 and higher are
available for download at www.biorad.com/bio-plex/standards
Bio-Plex Manager Software Version 5.0
Prepare System
1. Empty the waste bottle and make sure that there is sufficient sheath
fluid in the bottle or cube before proceeding.
2.
Turn on the reader, XY platform and HTF (if included). Allow the
system to warm up for 30 min.
3.
Select Start up
and follow the instructions to prepare the reader
to acquire data. If the system is idle for 4 hr without aquiring data, the
lasers will automatically turn off. To reset the 4-hour countdown, select
Warm up
and wait for the optics to reach operational temperature.
Calibrate With Low RP1 Target Value
Calibrate using Bio-Plex calibration beads and low RP1 target values on the
CAL2 bottle. This software version requires calibration only with the low
19
1.
Select Calibrate
and confirm that the default values for CAL1
and CAL2 are the same as the values on the Bio-Plex calibration
bead labels. Use the Bio-Plex low RP1 target value.
2.
Select OK and follow the instructions for CAL1 and CAL 2 calibration.
Prepare Protocol
Lot-specific assay panel protocols are available for download at
www.bio-rad.com/bio-plex/standards
If the desired protocol is not available for download, create a new protocol:
1.
Open a new protocol by selecting File, then New from the main
menu. Locate the steps at the left of the protocol menu.
2.
Select Step 1 (Describe Protocol) and enter information about the assay.
3.
Select Step 2 (Select Analytes) and choose the appropriate acute phase
panel, from the assay pull-down menu. If the panel is not listed, create
a new panel and select the “Bio-Plex Pro” assay type from the assay
pull-down menu. See the Bio-Plex Manager software instruction
manual for details.
NOTE: It is important to select the appropriate assay type in the assay
pull-down menu to ensure that data is acquired using the correct bead
map and gate settings.
4.
Choose the analytes to be selected for the assay from the available
list of targets. Note the targets will already be entered in the selected
view when using a downloaded protocol. In future runs, the selected
panel can be copied into the “available” view.
5.
Select Step 3 (Format Plate) and click on the Plate Formatting tab.
Select the number of replicates, the autofill direction, and click on
the
icon. Drag the cursor over all the wells that contain standards.
Repeat by replacing the
with
,
, and
to identify all the
wells that contain blanks, controls, and samples, respectively.
20
NOTE: If the preset protocol was downloaded from the web site, the
standards will already be added to the plate format. Make any
necessary changes to the plate layout to match your plate requirements.
Plate Formatting Example
6.
Select Step 4 (Enter Standards Info) to enter standards information.
Note that this information will already be entered when using a
download protocol. Otherwise:
a)
Deselect the box labeled “Same concentration values for all
analytes”. If analyzing a 5-plex assay, also deselect the box labeled
“Same units for all analytes”.
b)
Select the first analyte from the pull-down cell.
c)
Click on the Enter Automatically button and then select the
highest concentration value (typically S1).
d)
Enter the value for the highest concentration. The information is
included on the product insert provided with the assay.
e)
Enter the dilution factor (typically 4) and select Calculate. The
concentrations for each replicate point of the standard will be
populated for the selected analyte.
f)
Repeat steps 6b through 6e for each analyte in the assay.
21
7.
Select Step 5 (Enter Controls Info) to enter controls information. This
is where the concentration of the user-specified controls is entered
into the protocol.
a)
If necessary, deselect the box for same concentration values for
all analytes.
b)
Select an analyte from the pull-down cell.
c)
Enter the description and dilution information for each userspecified control.
d)
Repeat steps 7b and 7c for each additional analyte in the assay.
8.
Select Step 6 (Enter Sample Info) and enter sample information.
Remember to enter the appropriate dilution factor information.
9.
Select Step 7 (Run Protocol). Then:
22
a)
Choose 100 beads per region.
b)
In Advanced Settings, set the bead map to 25 region.
c)
In Advanced Settings, set the sample size to 50 µl.
d)
In Advanced Settings, confirm that the DD gate is set to 5,000
(low) and 32,000 (high).
e)
Save the protocol (.pbx) file.
Acquire Data
1. Shake the assay plate at 900 rpm for 30 sec immediately before
acquiring data. Failure to do so will result in increased read time due
to bead settling.
2.
Visually inspect the plate and ensure that the assay wells are filled with
buffer prior to placing the plate in the Bio-Plex microplate platform.
3.
Slowly remove the sealing tape and any plate cover before placing
the plate in the reader.
4.
Select the appropriate protocol (.pbx) file.
5.
Select Start and save the report (.rbx) file.
6.
Begin data acquisition.
NOTE: If acquiring data from more than one plate, empty the waste
bottle and refill the sheath bottle after each plate (if HTF not present).
Select Wash Between Plates
and follow the instructions for
fluidics maintenance. Then repeat the Prepare Protocol and
Acquire Data steps. We recommend using the Wash Between
Plates command after every plate run to reduce the possibility of
clogging the instrument.
7.
When data acquisition is complete, select Shut Down
the instructions.
and follow
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 Step 7 (Run Protocol). For
details, refer to the Bio-Plex Manager instruction manual.
23
Bio-Plex Manager Software Version 4.1 and 4.1.1
Prepare System
1. Empty the waste bottle and make sure that there is sufficient sheath
fluid in the bottle or cube before proceeding.
2.
Turn on the reader, XY platform and HTF (if included). Allow the
system to warm up for 30 min.
3.
Select Start up
and follow the instructions to prepare the reader
to acquire data. If the system is idle for 4 hr without aquiring data, the
lasers will automatically turn off. To reset the 4-hour countdown, select
Warm up
and wait for the optics to reach operational temperature.
Calibrate With Low RP1 Target Value
Calibrate using Bio-Plex calibration beads and the low RP1 target on the
CAL2 bottle. This software version requires the user to calibrate either on
the low or high RP1 target value. Daily calibration is recommended before
acquiring data.
1.
Select Calibrate
and confirm that the default values for CAL1 and
CAL2 are the same as the values on the Bio-Plex calibration bead
labels. Use the Bio-Plex low RP1 target value.
2.
Select OK and follow the instructions for CAL1 and CAL2 calibration.
Prepare Protocol
1. Open a new protocol by selecting File, then New from the main
menu. Locate the steps at the left of the protocol menu.
NOTE: To minimize protocol setup, previously prepared lot-specific
Bio-Plex Pro acute phase assay protocols in BPM 4.0 are available
for download at www.biorad.com/bio-plex/standards. The user
will need to select the 25 region bead map in Step 7 (Run Protocol).
2.
24
Select Step 1 (Describe Protocol) and enter information about the
assay.
3.
Select Step 2 (Select Analytes) and choose the appropriate acute
phase panel name, if available in the pull-down menu. If the panel
name is not available, create a new panel. See the Bio-Plex Manager
software instruction manual for details.
Choose the analytes to be selected from the available list of
targets. Note that the targets will already be entered in the selected
view when using a downloaded protocol. The selected panel
cannot be copied into the Available view and pull-down list with this
version of software, though it can be copied in Bio-Plex Manager
version 5.0.
4.
Select Step 3 (Format Plate) and click on the Plate Formatting tab.
Click on and drag the cursor over all the wells that contain standards.
Then click on and drag the cursor over the wells that contain blanks.
Repeat to identify all the wells that contain controls and to identify all
the wells that contain samples.
Plate Formatting Example
25
5.
6.
7.
26
Select Step 4 (Enter Standards Info) to enter standards information.
Note that this information will already be entered with the preset
download protocol.
a)
Select each analyte individually from the pull-down cell. Deselect
the box labeled “Same concentration values for all analytes”. If
analyzing a 5-plex assay, also deselect the box labeled “Same
units for all analytes”.
b)
Select the Enter Automatically option and then select the most
concentrated value as S1.
c)
Enter the concentration of S1 from the product insert provided
with the assay.
d)
Enter the dilution factor as 4 and select Calculate. The
standards information will be populated fro the selected analyte.
e)
Repeat steps 5a through 5d for each analyte in the assay.
Select Step 5 (Enter Controls Info) to enter controls information. This
is where the concentration of the user-specified controls is entered
into the protocol.
a)
Select each analyte individually from the pull-down cell. Deselect
the box for same concentration values for all analytes.
b)
Enter the description and dilution information for each userspecified control.
c)
Repeat steps 6a and 6b for each analyte in the assay.
Select Step 6 (Enter Sample Info) and enter sample information.
Acquire Data
1. Shake the assay plate at 900 rpm for 30 sec immediately before
acquiring data. Failure to do so will result in increased data
acquisition time due to bead settling.
2.
Visually inspect the plate and ensure that the assay wells are filled with
buffer prior to placing the plate in the Bio-Plex microplate platform.
3.
Slowly remove the sealing tape and any plate cover before placing
the plate in the reader.
4.
Select Step 7 (Run Protocol):
a)
Choose 100 beads per region.
b)
In Advanced Settings, set the bead map to 25 region.
c)
In Advanced Settings, set the sample size to 50 µl.
d)
In Advanced Settings, confirm that the DD gate are set to 5,000
(low) and 32,000 (high).
NOTE: These settings are already selected in preset
downloaded protocols.
e)
5.
Select Start and save the protocol (.pbx) file. Then follow the
instructions for data acquisition.
If acquiring data from ore than one plate, empty the waste bottle and
refill the sheath bottle after each plate (if HTF not present). Select Wash
Between Plates
and follow instructions for fluidics maintenance.
Then repeat the Prepare Protocol and Acquire Data steps.
NOTE: Use the Wash Between Plates command after every plate run
to reduce the possibility of clogging the instrument.
6.
When data acquisition is complete, select Shut Down
the instructions.
and follow
27
Reaquire Data
It is possible to acquire data from a well or plate a second time using the
Rerun/Recovery mode located below Start in Step 7 (Run Protocol). For
details, refer to the Bio-Plex Manager instruction manual.
Bio-Plex Manager Software Version 4.0
Bio-Plex Manager version 4.0 does not include the 25-bead map. Hence,
the 100-bead map is used. Follow the instructions above for Bio-Plex
Manager version 4.1 and 4.1.1 for preparing and calibrating the system.
Be careful to calibrate on Low RP1 and in Step 7 (Run Protocol) under
Advanced Settings, set the DD gates to 5,000 (low) and 32,000 (high).
Acquire Data
1. Shake the assay plate at 1,100 rpm for 30 sec immediately before
acquiring data. Failure to do so will result in increased data
acquisition time due to bead settling.
2.
Visually inspect the plate and ensure that the assay wells are filled with
buffer prior to placing the plate in the Bio-Plex microplate platform.
3.
Slowly remove the sealing tape and any plate cover before placing
the plate in the reader.
4.
Select Step 7 (Run Protocol):
a)
Choose 100 beads per region.
b)
In Advanced Settings, set the Bead Map to 100 region.
c)
In Advanced Settings, set the sample size to 50 µl.
d)
In Advanced Settings, confirm that the DD gates are set to 5,000
(low) and 32,000 (high).
NOTE: These settings are already selected in preset downloaded
protocols. If creating a new protocol, manually change the default
setting to reset the gates to 5,000 (low) and 32,000 (high).
e)
28
Select Start and save the report (.rbx) file. Then follow the
instructions for data acquisition.
5.
If acquiring data from more than one plate, empty the waste bottle
and refill the sheath bottle after each plate (if HTF not present). Select
Wash Between Plates
and follow the instructions for fluidic
maintenance. Then repeat the Prepare Protocol and Acquire Data
steps.
NOTE: Use the Wash Between Plates command after every plate run
to reduce the possibility of clogging the instrument.
6.
When data acquisition is complete, select Shut Down
the instructions.
and follow
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 Step 7 (Run Protocol). For
details, refer to the Bio-Plex Manager instruction manual.
Luminex xPONENT software
Luminex xPONENT software does not include the 25-bead map. Hence,
the 100-bead map is used. Refer to the detailed instructions in the
xPONENT software manual.
1.
Calibrate with Luminex MagPlex Classification Calibration
Microspheres and Luminex xMAP Reporter Calibration Microspheres
(sold by Luminex).
2.
Verify with xMAP MagPlex Classification Control Microspheres and
xMAP Control Microspheres (sold by Luminex).
3.
Create the acute phase panel protocol. Make sure to include the
correct bead region numbers and target names.
4.
The 100-region map should be selected.
5.
Set the gate settings at 10,000 (low) to 22,500 (high).
29
Section 11
Troubleshooting Guide
This troubleshooting guide addresses problems that may be encountered with
Bio-Plex Pro™ acute phase assays. If you experience any of the problems listed
below, review the possible causes and solutions provided. This will assist you in
resolving problems directly related to how the assay steps should be performed.
Poor assay performance may also be due to the Bio-Plex array reader. To
eliminate this possibility, we highly recommend use of the Bio-Plex validation kit.
This kit will validate all the key functions of the array reader and assist the user in
determining whether or not the array reader is functioning properly.
Possible Causes
Possible Solutions
High Inter-Assay %CV
Standards were not
reconstituted consistently
Incubate the reconstituted
standards for 30 min on ice. Always
be consistent with the incubation
time and temperature.
Reconstituted standards and
diluted samples were not stored
properly
Reconstituted standards and diluted
samples should be prepared on ice
as instructed. Prior to plating, the
reconstituted standards and diluted
samples should be equilibrated to
room temperature.
High Intra-Assay %CV
Bottom of filter plate not dry
30
Dry the bottom of the filter plate with
absorbent paper towel (preferably
lint-free) to prevent crosscontamination.
Possible Causes
Possible Solutions
Pipetting technique
Pipet carefully and slowly 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
were not equilibrated to room
temperature prior to plating
All reagents and assay components
should be equilibrated to room
temperature prior to plating.
Contamination with wash
buffer during wash steps
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. Also,
be sure that the microplate shaker
setting is not too high. Reduce the
microplate shaker speed to minimize
splashing.
Slow pipeting samples and
reagents across the plate
Sample pipeting across the entire
plate should take less than 4 min.
Reagent pipeting across the entire
plate should take less than 1 min.
31
Possible Causes
Possible Solutions
Low Bead Count
Miscalculation of bead dilution
Check your calculations and be
careful to add the correct volumes.
Beads clumped in multiplex
bead stock tube
Vortex for 15–20 sec at medium
speed before aliquoting beads.
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.
Did not shake filter plate enough
before incubation steps and prior
to reading
Shake the filter plate at 1,100 rpm
for 30 sec before incubation steps
and immediately before reading
the plate.
Reader is clogged
Refer to the troubleshooting guide
in the Bio-Plex hardware
instruction manual.
Low Signal or Poor Sensitivity
Standards reconstituted incorrectly
Follow the standard preparation
instructions carefully.
Detection antibody or
streptavidin-PE diluted incorrectly
Check your calculations and be
careful to add the correct volumes.
32
Possible Causes
Possible Solutions
High Background Signal
Incorrect buffer was used
(for example, assay buffer
used to dilute standards)
Use sample matrix or serum-based
diluent to dilute standards.
Spiked 0 pg/ml wells by mistake
Be careful when spiking standards.
Do not add any antigens in the
0 pg/ml (blank) point.
Streptavidin-PE incubated
too long
Follow the procedure incubation
time.
Poor Recovery
Expired Bio-Plex reagents were
used
Check that reagents have not
expired. Use new or unexpired
components.
Incorrect amounts of components
were added
Check your calculations and be
careful to add the correct volumes.
Microplate shaker set to an
incorrect speed
Check the microplate shaker speed
and use the recommended setting.
Setting the speed too high may
cause splashing and contamination.
Use the recommended plate shaker.
Pipetting technique
Pipet carefully and slowly 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.
33
Section 12
Safety Considerations
Eye protection and gloves are recommended while using this product.
Consult the MSDS for additional information.
34
xMAP, xPONENT and MagPlex are trademarks of Luminex Corp.
Costar is a trademark of Coming Costar Corporation. Eppendorf is a trademark of
Eppendorf-Netheler-Hinz GmbH. Luminex 100 and xMAP are trademarks of Luminex
Corporation. Multiscreen is a trademark of Millipore Corporation. Vortex-Genie is a trademark
of Scientific Industries, Inc.
By purchasing this kit, which contains fluorescent labeled microsphere beads authorized by
Luminex, you, the customer, acquire the right under Luminex's patent rights* to use this kit or
any portion of this kit, including without limitation the microsphere beads contained herein, only
with Luminex’s laser-based fluorescent analytical test instrumentation known under the name
of Luminex 100, for example as marketed by Bio-Rad Laboratories, Inc. in the Bio-Plex
system.
*Including, but not limited to US patent 5,981,180; 6,046,807; 6,057,107.
35
Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Dr.
Hercules, CA 94547 USA
1-800-424-6723 (in the US)
Bio-Rad
Laboratories, Inc.
Web site www.bio-rad.com USA 800 4BIORAD Australia 02 9914 2800
Austria 01 877 89 01 Belgium 09 385 55 11 Brazil 55 21 3237 9400
Canada 905 712 2771 China 86 21 6426 0808
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Greece 30 210 777 4396 Hong Kong 852 2789 3300
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Italy 39 02 216091 Japan 03 5811 6270 Korea 82 2 3473 4460
Mexico 55 5200 05 20 The Netherlands 0318 540666
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Portugal 351 21 472 7700 Russia 7 095 721 14 04
Singapore 65 6415 3188 South Africa 27 0861 246 723
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Taiwan 886 2 2578 7189/2578 7241 United Kingdom 020 8328 2000
Life Science
Group
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Rev A
Sig 1106
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