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Simplify
Multiplex Immunoassays with MultiBeadTM
Kits from Assay DesignsTM
MultiBead assays offer:
- Multiplexing capability - save time and
conserve precious samples
- Proprietary conjugation chemistry - detect low
abundant, large, and small molecule analytes with improved
sensitivity
- Quantitative data - obtain accurate results
with included standards
- Compatibility with common equipment -
conveniently run samples on dual-laser flow cytometers
Since 1992, Assay Designs has manufactured high quality antibodies,
proteins, and kits for the precise quantification of key large and small
molecule biomarkers. Assay Designs has now introduced MultiBead
multiplex immunoassays to simultaneously quantify the levels of up to 22
analytes in a single well and in various matrices, including serum,
plasma, urine, culture supernates, and cell lysates. Replacement of
traditional ELISA with multiplex immunoassays allows for simultaneous
evaluation of multiple analytes using minimal sample volume and offers
cost-effective and convenient quantification of biological markers. Best
of all, MultiBead assays do not require special instrumentation and work
on commercially available dual-laser flow cytometers, making access to
instrumentation convenient and economical.
MultiBead Frequently Asked Questions
Technology Overview
MultiBead assay technology utilizes the ability of commercially
available flow cytometers to separate latex beads covalently coupled to
antibodies on the basis of size and fluorescent intensity. MultiBead
assays can simultaneously quantify up to 22 analytes in a single well by
utilizing two sizes of beads (4.0 and 5.4 ?m) and 11 different bead
intensities. Each bead size is sub-fractioned into different populations
based on their inherent fluorescent intensity by a 670 nm filter, while
the analytes of interest are simultaneously quantified by a 575 nm
filter that measures fluorescence associated with phycoerythrin (PE)
conjugated detection antibody or analyte (Figure 1).
MultiBead assays are easy to perform and utilize a propriety conjugation
chemistry to covalently and efficiently couple the capture antibody to
the bead of interest. This improves assay sensitivity when compared to
beads that are coupled using traditional carboxy-conjugation methods. In
addition, unlike traditional immunoassays, MultiBead assays can be used
to simultaneously detect both large (immunometric assays) and small
(competitive immunoassays) analytes from a single sample (Figure 2).
Figure 1: Multiplexing Capability. Up to 22 distinct
analytes in various samples can be quantified by utilizing two sizes of
beads (4.0 and 5.4 µm) with 11 bead intensities each and the
fluorescence associated with the PE conjugate.
Figure 2: MultiBead Assay Procedure. MultiBead assays
can be used to simultaneously detect both large (immunometric assays)
and small (competitive immunoassays) analytes from a single sample.
After data collection, simply import the FCS or LMD data files into the
MultiBead Analysis Software to generate standard curves and determine
analyte concentrations.
Performance and Validation
To ensure accurate results, rigorous validation experiments are
performed to make certain that each bead specifically detects the
analyte of interest with no interference from other antibody-bead
populations or sample components (Figures 3-5).
Figure 3: High Specificity – 6-plex "Drop-in" Experiment.
Six immunometric bead sets were multiplexed in the presence of six PE
antibody conjugates. Individual cytokines (10,000 pg/mL) were then added
to show that the cytokine did not bind to the other 5 antibody pairs.
The 6-plex background PE fluorescence levels are shown in duplicate. The
data demonstrate that the antibody pairs chosen are specific and do not
cross-react with other cytokines.
Figure 4: High Specificity – "Drop-out" & "Drop-in" Experiments.
(A) Cytokine “Drop-out”: Multiplex analysis was performed with a panel
of cytokines, removing one of the cytokines at a time. When each
specific cytokine was removed, the corresponding fluorescence returned
to background levels, demonstrating antibody specificity.
(B) Eicosanoid “Drop-in”: A duplex assay was performed with TXB2 and
PGE2 antibody capture beads. Either TXB2 or PGE2 were then added to show
that each eicosanoid did not bind to the other bead. In a competitive
immunoassay, binding of analyte to the bead is reflected by a decrease
in fluorescence.
Figure 5: ELISA Comparison. The concentration of human IL-8 in various
matrices was quantified using Assay Designs MultiBead IL-8 Singleplex
kit (Cat. #985-004) and an ELISA kit from Supplier R. Similar data was
observed using the MultiBead Inflammation 8-plex panel.
| Sample A = Serum, Normal |
Sample D = Serum, Prostate Cancer |
| Sample B = Serum, Colon Cancer |
Sample E = Plasma, Prostate Cancer |
| Sample C = EDTA Plasma, Colon Cancer |
Sample F = Serum, Hypertension |
MultiBead Analysis Software
 The
MultiBead Analysis Software is available free of charge with the
purchase of any MultiBead kit to simplify and streamline data analysis.
This Assay Designs exclusive software allows users to generate standard
curves and determine sample concentrations for both immunometric and
competitive assays in a mutiplex bead format using FCS or LMD data
files. For details, please
download the software instruction manual.
The included MultiBead Analysis Software offers:
- Simplified data analysis – import FCS or LMD
files and analyze data using an easy-to-use interface
- Flexible tuning – adjust parameters and
settings to your needs
- Customized import and export – add your own
custom assay data and export to PDF or Excel
- High precision – optionally use triplicate data
series, 4PL or 5PL curve fit, and remove outliers
To request a CD
containing the MultiBead Analysis Software and accessory files (Cat.
#28-0001), please contact customer service at 1-877-423-4548 or
orders@assaydesigns.com (US customers only) or your
local distributor.
Free Software Downloads
|
MultiBead Analysis Software |
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Software Instruction Manual (2.63MB PDF) |
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| Sample
Data (ZIP Format) |
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Inflammation 8-Plex Kit File (ZIP Format) |
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Singleplex Kit Files (ZIP Format) |
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Ordering Information
MultiBead assays are available individually as singleplex kits or in a
pre-mixed and ready-to-use format that include beads, antibody
conjugates, sample dilution and wash buffers, and setup beads to correct
flow cytometer baseline values. For greater convenience, a buffer pack
is also available separately for use with individual singleplex kits. To
view our expanding portfolio of MultiBead assays, please visit this site
frequently for updates.
MultiBead assay types and kit contents:
| Kit Type |
Multiplex Panels |
Singleplex Kits |
Buffer Packs |
| Uses |
Ready-to-use kit for fast and convenient multiplex analysis |
For mix-and-matching or single analyte analysis |
Required for singleplex kits or when creating custom
multiplex assays |
| Kit Contents |
- Capture Bead cocktail
- Antibody Conjugate cocktail
- Standard cocktail
- Assay Buffer
- Wash Buffer
- Set-up beads
- Filter plate and tape
- Protocol
- Analysis Software
(free
download)
|
- Capture Bead
- Antibody Conjugate
- Standard
- Specification sheet
- Analysis Software (free
download)
*Buffer pack required |
- Assay Buffer
- Wash Buffer
- Set-up beads
- Filter plate and tape
- Protocol
|
Important Note:
- Singleplex kits can be combined with other compatible singleplex
MultiBead kits to create custom multiplex assays.
- Use of singleplex or custom multiplex assays
REQUIRES the purchase
of a compatible MultiBead Buffer Pack.
- Each MultiBead Buffer Pack contains sufficient reagents for one
96 well filter plate.
MultiBead Assays and Accessories
New MultiBead products are being introduced on a regular basis. To view
our up-to-date MultiBead product offering, please visit this site
frequently.
Click here to view abbreviations table
| Description |
Product Comments |
Sample Type |
Species Reactivity |
Catalog # |
Size |
|
Inflammation 8-Plex Panel |
Pre-mixed bead panel for simultaneous quantification of
IFN-gamma, IL-1beta, IL-4, IL-6, IL-8, PGE2,
TNF-alpha, and TXB2 via flow cytometry |
CS, P, S, U |
H |
980-001 |
1x96 well |
|
IFN-gamma Singleplex Kit |
Singleplex bead kit for quantification of human IFN-gamma
via flow cytometry |
CS, P, S, U |
H |
985-002 |
1x96 well |
|
IL-1beta Singleplex Kit |
Singleplex bead kit for quantification of human IL-1beta via
flow cytometry |
CS, P, S, U |
H |
985-003 |
1x96 well |
|
IL-4 Singleplex Kit |
Singleplex bead kit for quantification of human IL-4 via
flow cytometry |
CS, P, S, U |
H |
985-006 |
1x96 well |
|
IL-6 Singleplex Kit |
Singleplex bead kit for quantification of human IL-6 via
flow cytometry |
CS, P, S, U |
H |
985-005 |
1x96 well |
|
IL-8 Singleplex Kit |
Singleplex bead kit for quantification of human IL-8 via
flow cytometry |
CS, P, S, U |
H |
985-004 |
1x96 well |
|
PGE2 Singleplex Kit |
Singleplex bead kit for quantification of PGE2
via flow cytometry |
CS, P, S, U |
Species Independent |
985-008 |
1x96 well |
|
TNF-alpha Singleplex Kit |
Singleplex bead kit for quantification of human TNF-alpha
via flow cytometry. |
CS, P, S, U |
H |
985-001 |
1x96 well |
|
TXB2 Singleplex Kit |
Singleplex bead kit for quantification of TXB2
via flow cytometry. |
CS, P, S, U |
Species Independent |
985-007 |
1x96 well |
|
Extracellular Buffer Pack I |
Buffer pack intended for use with IFN-gamma, IL-1beta, IL-4,
IL-6, IL-8, PGE2, TNF-alpha, and TXB2
singleplex bead kits. |
N/A |
N/A |
987-001 |
1x96 well |
|
MultiBead Analysis Software |
Generate standard curves and determine sample concentrations
using FCS or LMD data files |
N/A |
N/A |
28-0001 |
1 CD |
Custom MultiBead Assay Services
The Assay Designs research and development team possesses extensive
immunoassay development experience gained through greater than 16 years
developing commercial immunoanalytical assays utilizing colorimetric,
fluorescent, chemiluminescent, and fluorescence polarization detection
systems. Our assay development experience includes a wide range of
analytes from small molecules to large proteins and their various post
translational modifications. For more information about custom MultiBead
assay development, please visit
www.assaydesigns.com/customservices.
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