Bio-Rad Bio-Plex Pro Human Th17 Cytokine Assays User Manual
Bio-Plex Pro™ Human
Th17 Cytokine 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
Important Considerations 7
Detailed Instructions 7
1. Plan Plate Layout 8
2. Prepare Instrument 9
3. Prepare Wash Method 10
4. Prepare Standards and Controls 11
5. Prepare Samples 13
6. Prepare Coupled Beads 16
7. Run Assay 17
8. Read Plate 22
Troubleshooting Guide 29
Plate Layout Template 34
Calculation Worksheet 35
Safety Considerations 37
Legal Notices 37
Ordering Information 38
Introduction
Bio-Plex Pro™ Human Th17 Cytokine Assays
Bio-Plex Pro human Th17 cytokine assays are designed to meet the needs
of the most demanding preclinical and clinical research environments.
These magnetic bead-based multiplex immunoassays include a
comprehensive blend of soluble biomarkers involved in the T-helper cell
type 17 immune response pathway, a key driver of autoimmune diseases,
as well as the body’s response to microbial infection. With diluents
optimized for enhanced performance in clinical matrices, a fast protocol,
and a flexible kit format, the Human Th17 cytokine assays promise to make
multiplexing easier and more reliable than ever.
Multiplexing with Bio-Plex Pro Assays
Bio-Plex Pro assays enable researchers to quantify multiple protein
biomarkers in a single well of a 96-well plate in just 3 to 4 hr. These
robust immunoassays require as little as 12.5 µl of serum or plasma,
or 50 µl of other biological fluid. The use of magnetic (MagPlex) beads
allows researchers to automate wash steps on a Bio-Plex Pro (or similar)
wash station. Magnetic separation offers greater convenience and
reproducibility compared to vacuum filtration.
For more information please visit www.bio-rad.com/bio-plex
1
Principle
Technology
The Bio-Plex
elements of xMAP technology:
• Fluorescentlydyedmicrospheres(alsocalledbeads),eachwithadistinct
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
•Adedicatedowcytometerwithtwolasersandassociatedopticsto
measure the different molecules bound to the surface of the beads
• Ahigh-speeddigitalsignalprocessorthatefcientlymanagesthe
fluorescence data
Assay Format
Bio-Plex Pro
magnetic beads. The assay principle is similar to that of a sandwich
ELISA (Figure 1). Capture antibodies directed against the desired
biomarker are covalently coupled to the beads. Coupled beads react
with the sample containing the biomarker of interest. After a series of
washes to remove unbound protein, a biotinylated detection antibody
is added to create a sandwich complex. The final detection complex is
formed with the addition of streptavidin-phycoerythrin (SA-PE) conjugate.
Phycoerythrin serves as a fluorescent indicator, or reporter.
®
suspension array system is built around the three core
™
assays are essentially immunoassays formatted on
2
BIOMARKER
OF INTEREST
STREPTAVIDIN
MAGNETIC BEAD
CAPTURE
ANTIBODY
Figure 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 (pg/mL). The concentration of
analyte bound to each bead is proportional to the MFI of reporter signal.
3
Kit Contents and Storage
Reagents Supplied
Bio-Plex Pro
™
human Th17 cytokine 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 premixed and custom x-Plex™ kits.
**
Component
Standard diluent HB
Sample diluent HB
Assay buffer
Wash buffer
Detection antibody diluent
Streptavidin-PE (100x)
Assay plate (96-well flat-bottom plate or filter plate)
Sealing tape
Assay Quick Guide
Coupled magnetic beads (20x)
Detection antibodies (20x)
Standard
Quality Control High*
Quality Control Low
* Only premixed Th17 panels and custom Th17 x-Plex kits include quality controls.
** Volumes shown are approximate. Quantities in Express assays will vary.
*
Quantity
10 ml
8 ml
50 ml
200 ml
5 ml
1 tube
1 plate
1 pack of 4
1 booklet
1 tube
1 tube
1 vial
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 6 months from the date of purchase
when stored as specified.
4
Table 2. Recommended materials.
Item
Bio-Plex Pro Assays Quick Guide 1
Ordering Information
Item #10022581 (download at
www.bio-rad.com/bio-plex)
®
Bio-Plex
Bio-Plex validation kit
200 system or Luminex system with HTF
Bio-Rad catalog #171-000205
Bio-Rad catalog #171-203001
Note: the validation kit should be run monthly to ensure
performance of fluidics and optics systems
Bio-Plex calibration kit
Bio-Rad catalog #171-203060
Note: the calibration kit should be run either daily or
whenever the instrument is to be used 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 (40, 96-well plates)
Bio-Rad catalog #171-025001
For magnetic separation on the Bio-Plex Pro wash station
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)
™
Bio-Rad Aurum
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
vacuum manifold
Bio-Rad catalog #732-6470
Bio-Rad catalog #166-0610
VistaLab catalog #3054-1002
or
VistaLab catalog #3054-1004
VistaLab catalog #3054-1006
Pall Life Sciences
catalog #4187
Bio-Rad catalog #171-304502
Other: 15 ml polypropylene tubes for reagent dilutions. Calibrated pipets (multichannel pipets
are optimal for this application) and 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 beads
Wash 2x
Add standards, samples, controls, 1 hr
Wash 3x
Add detection antibody, 30 min
Wash 3x
Add streptavidin-PE, 10 min
Wash 3x
Resuspend
Acquire data
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 8).
Assay Procedures
Please pay close attention to vortexing, shaking, and incubation times,
and Bio-Plex
®
reader PMT (RP1) setting, as these have been optimized
specifically for each assay panel.
Assay Quick Guide
Each assay kit comes complete with a printed Bio-Plex Pro
™
Assay Quick
Guide (item #1002258), 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 and Multiplexing Compatibility
n
Bead regions for all analytes are listed in the Read Plate section.
n
For optimum performance of the human Th17 cytokine assays, mixing
of Th17 analytes with other Bio-Plex assay panels or reagent kits is not
recommended. For example:
a. Overlapping analytes in different antigen standards should not be mixed
b. Bio-Plex Pro reagent kit II contains diluents that have not been fully
validated in other assays
In each case, standard curves and sample values may be inaccurate.
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 34, or the Plate
Formatting tab in Bio-Plex Manager
in Figure 2, with all conditions run in duplicate.
1. Assign standards to columns 1 and 2, with the highest
concentration in row A and the lowest concentration in row H.
™
. A suggested plate layout is shown
2. Assign the blank to wells A3 and A4. The blank should consist of your
chosen standard diluent. Note that Bio-Plex Manager
™
automatically
subtracts the Blank (B) FI value from all other assay wells.
3. User-specified controls, as well as the quality controls supplied in
premixed Th17 cytokine kits, are assigned to wells in colums 3 and 4.
4. The remainder of the plate is available for samples.
5. Once the total number of wells is known, you can calculate the
required volumes of beads, detection antibody, and streptavidin-PE.
Use Tables 6, 9, and 11, respectively, or the Calculation Worksheet
on pages 35–36.
Fig. 2. Suggested plate layout. For detailed instructions on
plate formatting in Bio-Plex Manager, see Section 8. Legend
S Standards
B Blank
X Samples
C Controls
8
2. Prepare Instrument
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 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.
2. Select OK and follow the software prompts for step-by-step
instructions for CAL1 and CAL2 calibration.
NOTE! In Bio-Plex Manager v6.1 and higher, startup, warm up and calibrate
can be performed together by selecting the start up and calibrate icon.
9
3. Prepare Wash Method
There are two methods of performing wash steps in the assay procedure:
magnetic separation and vacuum filtration. Compatible wash stations and
plate types are shown in the table below.
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)
Magnetic Separation Using 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.
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
(item #10023087).
™
Flat-bottom plate
Vacuum Filtration Using 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 ul liquid should take 3–4 sec to clear the well. For more detailed
instructions, refer to item #10005042.
10
4. Prepare Standards and Controls
General Instructions
• Itisessentialtopreparestandardsandqualitycontrols(ifincluded)
exactly as described in this section. Incorrect preparation may lead to
low signal or variable measurements from plate to plate
• Theproductdatasheetorpeel-offstickerprovidedwiththestandards
lists the most concentrated point on the standard curve (S1). Enter this
information into Bio-Plex Manager
Using the Quality Controls (optional)
Two-level quality controls (high and low) are included with premixed
Th17 cytokine panels and custom x-Plex kits. Their use is intended for
monitoring the day-to-day quality of assay results.
Selecting a Diluent for Standards and Controls
Refer to Table 4 for recommended diluents based on different sample types.
™
software as instructed in Section 8
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
®
standard diluent HB. 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 standards and controls.
Diluent for Standards
Sample Type and Controls* Add BSA
Serum and plasma Standard diluent HB None
Culture media, with serum Culture medium None
Culture media, serum-free Culture medium To 0.5% final**
Lavage, sputum, other fluids Sample diluent HB To 0.5% final**
Lysate Sample diluent HB To 0.5% final**
* If using diluents other than standard diluent HB, users must establish their own control ranges.
** At least 0.5% final BSA is recommended to stabilize analytes and reduce absorption to labware.
11
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