Bio-Rad Bio-Plex Pro Human Isotyping Assays User Manual
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
Introduction 1
Principle 2
Kit Contents and Storage 4
Recommended Materials 5
Assay Workflow 6
lmportant Considerations 7
Detailed Instructions 7
1.1. Plan Plate Layout 8
2.2. Prepare Instrument 9
3.3. Prepare Wash Method 10
4.4. Prepare Wash Buffer 11
5.5. Prepare Standards and Controls 11
6. Prepare Samples 14
7.7. Prepare Coupled Beads 16
8.8. Run Assay 17
9.9. Read Plate 21
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 l 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
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
n
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
n
A high-speed digital signal processor that efficiently manages the
fluorescence data
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
Fig. 1. Bio-Plex sandwich immunoassay.
Biotinylated
Detection
Antibody
Phycoerythrin
Fluorescent
Reporter
Data Acquisition and Analysis
Data from the reactions are acquired using a Bio-Plex system or similar
Luminex-based reader. When a multiplex assay suspension is drawn into
the Bio-Plex 200 reader, for example, a red (635 nm) laser illuminates the
fluorescent dyes within each bead to provide bead classification and thus
assay identification. At the same time, a green (532 nm) laser excites PE
to generate a reporter signal, which is detected by a photomultiplier tube
(PMT). A high-speed digital processor manages data output, and Bio-Plex
Manager
™
software presents data as median fluorescence intensity (MFI) as
well as concentration. The concentration of analyte bound to each bead is
proportional to the MFI of 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
Isotyping diluent
Assay buffer
Wash buffer (10x)
Detection antibody diluent
Streptavidin-PE (100x)
Assay plate (96-well flat bottom plate)
Sealing tape
Assay quick guide
Product data sheet
Coupled magnetic beads (20x)
Detection antibodies (20x)
Standard
Quality Control
* Volumes shown are approximate.
1 bottle, 180 ml
1 bottle, 50 ml
1 bottle, 60 ml
1 bottle, 5 ml
Quantity
1 tube
1 plate
1 pack of 4
1 booklet
1
1 tube
1 tube
1 vial
1 vial
*
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
Bio-Plex® 200 system or Luminex system with HTF
Bio-Plex validation kit
Ordering Information
Bio-Rad catalog #171-000205
Bio-Rad catalog #171-203001
Note: Run the validation kit monthly to ensure optimal
performance of fluidics and optics systems
Bio-Plex calibration kit
Bio-Rad catalog #171-203060
Note: Run the calibration kit daily to standardize
fluorescence signal
Bio-Plex Pro wash station
Bio-Rad catalog #300-34376
For use with magnetic bead–based assays only
Bio-Plex Pro II wash station
Bio-Rad catalog #300-34377
For use with both polystyrene (nonmagnetic) and magnetic
bead–based assays
Bio-Plex handheld magnetic washer
Bio-Rad catalog #170-20100
For use with magnetic bead–based assays only
Bio-Plex Pro flat bottom plates (forty 96-well plates)
Bio-Rad catalog #171-025001
For magnetic separation on the Bio-Plex Pro wash station
®
Titertube
For preparing replicate standards, samples, and controls
micro test tubes
Bio-Rad catalog #223-9390
prior to loading the plate
Microtiter plate shaker
IKA MTS 2/4 shaker for 2 or 4 microplates
IKA catalog #320-8000
or
Barnstead/Lab-Line Model 4625 plate
VWR catalog #57019-600
shaker (or equivalent capable of 300–1,100 rpm)
®
Aurum™ vacuum manifold
Bio-Rad
Bio-Rad catalog #732-6470
For vacuum filtration
BR-2000 vortexer
Reagent reservoirs, 25 ml
For capture beads and detection antibodies
Reagent reservoir, 50 ml (for reagents and buffers)
Pall Life Science Acrodisc: 25 mm PF syringe filter
(0.8/0.2 µm Supor membrane)
Filter plate, 1 x 96 with clear plastic lid and tray
Bio-Rad catalog #166-0610
VistaLab catalog #3054-1002
or
VistaLab catalog #3054-1004
VistaLab catalog #3054-1006
Pall Life Sciences
catalog #4187
Bio-Rad catalog #171-304502
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 lter 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 (IgG
, IgG2, IgG3, and IgG4).
1
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
7
™
and Luminex xPONENT software.
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
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
™
software. A suggested plate layout is
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
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.
®
MAGPIX™ reader, consult their respective
™
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
for 4 hr without acquiring data, the lasers will automatically turn off.
To reset the 4-hr countdown, select Warm up
lasers/optics to reach operational temperature.
and follow the instructions. If the system is idle
and wait for the
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
Vacuum filtration Bio-Plex Pro II (use VAC programs) Filter plate
Vacuum manifold (manual)
Setting up the Bio-Plex Pro or Bio-Plex Pro II
Wash Station
The wash station 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.
™
Flat bottom plate
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
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