Archives of Physiology and Biochemistry, 2012; 118(4): 192–196

© 2012 Informa UK, Ltd.

ISSN 1381-3455 print/ISSN 1744-4160 online
DOI: 10.3109/13813455.2012.705301

ORIGINAL ARTICLE

Bio-Rad’s Bio-Plex® suspension array system, xMAP

technology overview
Brett Houser

Bio-Rad Laboratories, Inc., Hercules, California, USA

Abstract
The Bio-Plex® system utilizes xMAP technology to permit the multiplexing of up to 100 different analytes. Multiplex
analysis gives researchers the ability to look at analytes simultaneously providing more information from less sample
volume in less time than traditional immunoassay methods. Similar to ELISA, xMAP utilizes an antibody sandwich
for detection but differs from ELISA in capture substrate and detection method. Rather than a flat surface, Bio-Plex®
assays make use of differentially detectable bead sets as a substrate capturing analytes in solution and employs
fluorescent methods for detection. These bead sets identify the analytes and detection antibodies are used to
measure the quantity of analyte. The use of differentially detectable beads enables the simultaneous identification
and quantification of many analytes in the same sample.
Keywords:  Immunoassay, multiplex detection, ELISA, Bio-Plex®, xMAP, Luminex

Background antibodies and chemiluminescence detection resulted in

ELISA assays with sensitivity that exceeds that of radiola-
For more than 50 years, immunoassays have allowed
bels. Today, key advantages of ELISA are its ease of use,
sensitive and highly specific detection of analytes of
flexibility and low cost. The impact of immunoassays on
interest in biological samples in life science research and
life science research and clinical diagnostics has been
clinical diagnostics. Immunoassays provide informa-
enormous, with almost 10,000 studies published per
tion to researchers on the roles proteins and other bio-
year that include the terms “enzyme immunoassay” and
molecules play in a myriad of biological processes and
“enzyme-linked immunoassay” (Lequin 2005).
diseases. The first immunoassay was developed by Yalow
and Berson (1960), who received the Nobel Prize for their
efforts to measure insulin levels. These initial assays used
Multiplex detection and measurement using
radiolabels for detection. The radioimmunoassay (RIA)
xMAP technology
would remain the standard for detection of bio-analytes
for more than 10 years because of its extraordinary sen- The Bio-Plex® suspension array system utilizes xMAP
sitivity, in spite of the health risks and disposal issues technology, licensed from Luminex Corp., to permit
posed by the use of radioisotopes. The search for a suit- the multiplexing of up to 100 different assays within a
able alternative to the RIA led to the development of the single sample. This technique involves 100 distinctly
enzyme-linked immunosorbent assay (ELISA) in the coloured bead sets created by the use of two fluorescent
early 1970s (Engvall and Perlmann 1971, Van Weeman dyes at distinct ratios. Beads are conjugated with a
and Schuurs 1971). ELISA uses an enzymatic reaction reagent specific to a particular bioassay. The reagents
as the basis of detection, rather than a radioactive sig- may include antigens, antibodies, oligonucleotides,
nal. While early versions did not rival the sensitivity of enzyme substrates or receptors. The technology makes
the RIA, the development of highly specific monoclonal use of multiple assays whereby one antibody to a specific

Address for Correspondence:  Brett Houser, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547, USA 510–741–5613.
E-mail: [email protected]
(Received 03 May 2012; revised 15 June 2012; accepted 19 June 2012)

192
 Bio-Rad’s Bio-Plex® suspension array system, xMAP technology overview 193
analyte is attached to a set of beads with the same colour is washed to remove unbound materials, followed by incu-
and the second antibody is used to quantify the bound bation with biotinylated detection antibodies. After wash-
antigen. The use of different coloured beads enables ing away the unbound biotinylated antibodies, the beads
the simultaneous detection of many other analytes in are incubated with a reporter streptavidin-phycoerythrin
the same sample. Imaging or laser excitation is then conjugate (SA-PE). Following removal of excess SA-PE, the
used to determine the different assays by bead colours, beads are passed through the array reader, which measures
and determine analyte concentration by measuring the the fluorescence of the bound SA-PE (Figure 4).
reporter dye fluorescence (Figure 1). The substrate for the antibody sandwich is the bead.
For laser excitation detection (Bio-Plex 200 and Bio- Bead characteristics define instrument compatibility
Plex 3D) the contents of each microplate well are drawn and workflow and can be classified into two categories,
into the array reader and precision fluidics align the non-magnetic and magnetic. The non-magnetic beads
beads in single file through a flow cell, where two lasers are smaller in size (5.6 μm and are used with the vacuum
excite the beads individually. The red classification laser workflow; they are not compatible with instruments that
excites the dyes in each bead, identifying its spectral utilize magnets for imaging purposes. Non-magnetic
address. The green reporter laser excites the reporter beads require filter plates and vacuum filtration to wash
molecule associated with the bead, which allows quan- the beads. Magnetic beads are coated with magnetite
titation of the captured analyte. High-speed digital signal and are therefore larger in size (6.5 μm); they can be used
processors and software record the fluorescent signals with the magnetic workflow as well as vacuum workflows.
simultaneously for each bead, translating the signals into These magnetic beads are compatible with all currently
data for each bead-based assay (Figure 2). available life science instruments from any Luminex
The Bio-Plex MAGPIX system employs low-cost light- partner. In the magnetic workflow, the beads are washed
emitting diodes (LEDs) and a charge-coupled device in the well with dispense and aspiration washing.
(CCD) imager to illuminate and image immobilized
magnetic beads Figure 3. Unlike flow-based systems
that quantitate bead events individually, the Bio-Plex
About the beads
MAGPIX system reads all of the beads at once. This xMAP assays may contain non-magnetic or magnetic
dependable design is not only very robust, but also beads as substrates. Magnetic COOH beads are the new-
reduces instrument preparation time compared to flow- est core components of xMAP assays. They are unique
based design – all the while giving very comparable data. in that they exhibit both fluorescent and magnetic
properties. The beads are stained with a fluorescent dye
formulation proprietary to Luminex (Figure 5). The stain-
Workflow overview ing process involves swelling the bead particles in a dye
Similar to ELISA, a majority of assays are designed accord- containing solvent allowing the dye molecules to infuse
ing to a capture sandwich immunoassay format. Briefly, the the coating or the polymer layer. Removal of the solvent
capture antibody-coupled beads are first incubated with in a subsequent step shrinks the beads and traps the dye
antigen standards or samples for a specific time. The plate molecules within the bead particles.

Figure 1. Multiplex immunoassay technology. Beads are colored internally with two different fluorescent dyes (red and infrared).
Ten different concentrations of red and infrared dyes are used to generate 100 distinct bead regions. Each bead region is conjugated to a
specific target analyte.

© 2012 Informa UK, Ltd.

194  B. Houser

Figure 2.  Data acquisition and reduction. Dyed beads are pushed through a detection chamber in a single file. The red classification laser
(635 nm) interrogates internal dye to identify bead regions. The green reporter laser (532 nm) interrogates fluorescent reporter to measure
analyte concentration.

Figure 3.  Immobilized magnetic beads illuminated with LED’s and imaged using CCD technology.

Figure 4.  xMAP workflow.

The use of magnetic beads confers a number of ben- Advantages of bead based multiplex
efits, including ease of separation and suitability for detection over conventional methods
automation. Once coated with ligand molecules, the
magnetic beads are useful for the capture and separation ELISA
of a variety of targets. Unwanted sample constituents The xMAP technology is frequently compared to the tra-
such as large or fibrous particulates or a viscous sample ditional ELISA technique, which is limited by its ability
matrix may be washed away following a simple magnetic to measure only a single antigen. The inability of tradi-
separation step. Overall, the highly efficient magnetic tional ELISA to multiplex presents additional limitations
separation eliminates potentially interfering molecules, in sampling volume, cost and labour (Table 1). While the
allowing sensitive detection of targets (Figure 6). basic concepts of detection are similar between ELISA

 Archives of Physiology and Biochemistry

 Bio-Rad’s Bio-Plex® suspension array system, xMAP technology overview 195

Figure 7.  Visualization of sample relationships by analyte using

Bio-Plex® Data Pro Software.
Figure 5.  Magnetic bead architecture.
Western blot
Bio-Plex cell signalling assays can be used in place of
classic western blot methods. For example investigat-
ing five proteins in 12 samples by western blot would
require five gels and blots, 5 times more samples
(125 ul vs. 25 ul) and about 5 hours longer to obtain
results (Table 2).

Multiplex assay applications

The use of xMAP technology is well established with
over 7000 peer-reviewed publications to date. Bio-Rad’s
Bio-Plex® assays are available for many classes of bio-
molecules and species including cytokines and growth
factors, specialized disease state panels for cancer, acute
phase, immune response and diabetes. Assays are avail-
Figure 6.  Hands-free washing and separation of magnetic beads. able in several flexible configurations. Commercially
available kits are typically developed with optimized pro-
Table 1.  Side-by-side comparison: Analysing 27 cytokines in 80 tocols. Alternatively, tools and individual components
samples. are available for researchers to develop their own assays.
ELISA Bio-Plex
Number of cytokines 27 27 Data acquisition and analysis via Bio-Plex® software
Number of samples 80 80 An integral component of multiplex immunoassay
Total data points 2160 2160 experiments, Bio-Plex® software provides an intui-
Number of 96-well 27 1 tive interface for input of sample properties, allowing
plates researchers to handle clinical information from large
Data points per plate 80 2160 cohorts of samples and tools for analysis of complex pro-
Total time required >60 hours 3 hours
teomic data generated by the Bio-Plex® system.
Sample volume Serum or plasma, Serum or plasma,
Biomarker discovery driven data analysis tools include
>1 ml 12.5 μl
*Cell culture Cell culture multiple modes of spectral visualization. Differentially
supernatant, >1 ml* supernatant, 50 μl expressed proteins are sought through univariate analy-
Assay range 3 to 4 logs dynamic 5 to 6 logs dynamic ses, including Mann-Whitney and Kruskal-Wallis tests,
range range receiver operating characteristic curves, and dot plots.
Note: *Based on 50 μl/well sampling volume. Relationships between samples may be further explored
with Bio-Plex® Data Pro software (Figure 7).
and xMAP technology, there are some important dif-
ferences that centre on the capture antibody support. Definition of quality
Unlike traditional ELISA, xMAP capture antibodies are Assay quality is defined by a series of experiments
covalently attached to a bead surface, effectively allowing establishing performance characteristics of the assay,
for a greater surface area as well as a matrix or free solu- such as accuracy, precision, specificity and sensitivity.
tion/liquid environment to react with the analytes. The Taking into account that an assay should provide reliable
suspended beads allow for assay flexibility in a singleplex results, the concept of assay quality is generally viewed in
or multiplex format. a broader perspective. Design of the assay workflow, the

© 2012 Informa UK, Ltd.

196  B. Houser
Table 2.  Side-by-side comparison: Analysing 5 phosphoproteins in 12 samples.

Western Blot Bio-Plex Assays

Number of phosphoproteins 5 5
Number of samples 12 12
Total data points 60 60
Number of gels or 5 1
96-well plates

Data points per gel or plate 12 60

Total time required ~8 hr + one overnight incubation 3 hr + one overnight incubation
Sample volume 125 µl 25 µl
Dynamic range Depends on detection method 50-900 µg/ml total protein
Sensitivity* 30,000

20,000

MFI
5
10 2.5 1.25 0.63 0.31 0.16 0.08 0.04 0.02 0.01 .005
0 10,000
Total protein, µg
Western blot of total HSP27 (COS-7 cell lysate) 0
5 3 1
10 2.5 1.25 0.6 0.3 0.16 0.08 0.04 0.02 0.01 .005
0
Total protein, µg
Bio-Plex total HSP27 assay (COS-7 cell lysate)

sample matrices to be tested, and the utility of the test Over the years, the power and ease-of-use of Bio-
results should also be taken into consideration. Plex® software has delivered dominant market share in
Immunoassay standard curves are inherently non- xMAP instruments; as a result, most xMAP researchers
linear. As such, a minimum of six non-zero standard use Bio-Plex® to get the answers they need.
points assayed in duplicate is recommended to ensure
the robustness of an assay. The concentration–response Declaration of interest
relationship is most often fitted to a 4- or 5-parameter
logistic model. The author is an employee of Bio-Rad Laboratories.
Bio-Rad, Bio-Plex® multiplex immunoassays are
subject to rigorous quality measures. The assays are References
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immunosorbent assay (ELISA). Clin Chem 51(12):2415–18.
ners to license this dynamic technology. The Bio-Plex®
Van Weeman BK, Schuurs AH. (1971). Immunoassay using antigen-
product family provides a complete solution: multiplex enzyme conjugates. FEBS Lett 15(3):232–6.
assays, the Luminex instruments, and proprietary inno- Yalow RS, Berson SA. (1960). Immunoassay of endogenous plasma
vative software for data generation and analysis. insulin in man. J Clin Invest 39:1157–75.

 Archives of Physiology and Biochemistry