Composite Profiling of Angiogenic Factors Using Bio-Plex Pro™
Human Cancer Biomarker Panel 1
Richard Zimmerman, Tim Hamilton, Li Ma, and Vinita Gupta,
Life Science Group, Bio-Rad Laboratories, Inc., 2000 Alfred Nobel Drive, Hercules, CA 94547 USA
Angiogenesis is a fundamental process required for multiple
physiological and pathological events. It is also a hallmark of
over 50 different disease states, including cancer, rheumatoid
arthritis, cardiovascular diseases, diabetes, and psoriasis
(Staton et al. 2004, 2009). Methods developed to study these
diseases are important tools for testing potential therapeutics.
These methods include both in vivo and in vitro assays.
In vivo assays are considered the most informative because
of the complex nature of vascular responses to test reagents.
However, these assays are often costly and laborious.
In contrast, in vitro assays can be carried out expeditiously,
are less expensive, and are easier to interpret. Often, these
in vitro assays provide maximum benefits when developed
as multivariate index assays where the data of multiple
assays yield a composite profile of clinically relevant
Using Luminex xMAP technology (Dale et al. 2008 and bulletin
5404), we have developed a multiplex Bio-Plex Pro human
cancer biomarker panel that employs a magnetic bead-based
workflow to measure angiogenesis biomarkers in diverse
matrices including serum, plasma, cell culture supernatant,
and many other sample types. In combination with the
Bio-Plex® suspension array and the Bio-Plex Pro wash
station, the multiplexing feature makes it possible to quantify
multiple angiogenesis targets in a single well of a 96-well
microplate in just three hours, using as little as 12.5 μl of
serum or plasma.
The panel of 16 markers comprises sEGFR, FGF-basic,
follistatin, G-CSF, HGF, sHER2/neu, sIL-6Ra, leptin,
osteopontin, PECAM-1, PDGF-AB/BB, prolactin, SCF,
sTIE-2, sVEGFR-1, and sVEGFR-2 (Table 1). These markers
were selected because of their direct relevance to tumor-
associated angiogenesis (Bridges et al. 2011). However, the
panel is relevant to other disease-related processes such
as metastasis, cell proliferation, cell adhesion, apoptosis,
The assays were validated on human serum, plasma, and
cell culture matrices. Validation criteria include working assay
range (LLOQ/ULOQ), sensitivity (LOD), intra- and inter-assay
precision, specificity and cross-reactivity, linearity of dilution,
and parallelism to evaluate robustness in the key sample
matrices mentioned above.
The principle of these 96-well plate-formatted bead-based
assays is similar to a capture sandwich immunoassay (Figure 1).
The capture antibody–coupled beads were first incubated
with antigen standards or samples followed by incubation
with biotinylated detection antibodies. After washing away
the unbound biotinylated antibodies, the beads were
incubated with a reporter streptavidin-phycoerythrin
(SA-PE) conjugate. Following removal of excess SA-PE,
the beads were passed through the Bio-Plex array reader
(Bio-Plex 200 system), which measures the fluorescence of
the bead and of the bound SA-PE. Assay incubations were
performed at room temperature according to the settings
shown in Table 2. All washes were performed using a
Bio-Plex Pro wash station. Data acquisition was performed
using Bio-Plex Manager™ 6.0 at a low PMT setting.
Assay Performance Characteristics
The angiogenesis assays were evaluated according to the
following parameters: assay sensitivity, precision, accuracy, assay
working range, cross-reactivity, matrix effects, and validation with
biological samples. Assay sensitivity, defined as limit of detection
(LOD), was evaluated by adding two standard deviations to the
mean median fluorescence intensity (MFI) of ten zero standard
replicates. With the exception of osteopontin and PECAM-1,
all the targets reported concentrations below 10 pg/ml in both
serum-based standard diluent and RPMI cell culture media
(Table 3). Osteopontin recorded 56.4 pg/ml in standard diluent.
PECAM-1 recorded 24.4 pg/ml and 39.6 pg/ml in standard diluent
and RPMl, respectively.
Assay reproducibility evaluates both intra-assay %CV and
inter-assay %CV. Intra-assay %CV assesses variation among
the replicates within the assay. Inter-assay %CV measures the
variability across three independent assays. Intra-assay %CV
was calculated from the median fluorescent intensity (MFI) of all
three replicates at each standard antigen dilution point from a
representative assay in serum-based standard diluent and RPMI
matrices. All the targets demonstrated <10 %CV in both serum
and RPMI matrices. Inter-assay %CV for multiplex assays was
determined from three independent plates using the observed
concentration of a 6-level spike control and the observed
concentration of within assay range standard points. All the
targets demonstrated <15 %CV in both serum and RPMI
matrices (Table 4).
Notes: The mean intra-assay %CV values were derived from 16 multiplex and singleplex assays in a serum matrix and RPMI cell culture medium, and from
16 multiplex assays in RPMI cell culture medium. For inter-assay %CV (derived in multiplex format), the mean spike control %CV was derived from 6 serially diluted
concentration levels. The mean standard curve %CV was derived from working assay range standard points.
Assay accuracy (also defined as recovery) was calculated
as the percentage of the observed concentration value of a
target antigen relative to the expected value. This parameter
was evaluated using standard points and spiked controls
in both multiplex and singleplex configurations, and in
both serum and RPMI. Overall, the standard recovery was
comparable in both matrices, with most targets recovering
80–120% within assay working ranges (Table 5). The recovery
in both single- and multiplex settings was also comparable
(data not shown).
Assay working range is defined as the range between the
lowest level of quantification (LLOQ) and the upper level of
quantification (ULOQ) in which an assay is both precise and
accurate. The ranges of these assays were determined for
both serum and RPMI cell culture medium. Table 6 lists the
assay ranges in multiplex and singleplex assays in a serum
matrix and RPMI cell culture medium supplemented with
10% FBS. The reproducibility of these ranges is dictated by
the overall precision in preparing the assay reagents.
Assay specificity was examined by performing single-antigen
and single-detection cross-reactivity studies. The single-
antigen study evaluates the specificity of a capture antibody.
This was conducted by testing an individual antigen in
the presence of multiplexed capture beads and detection
antibodies. The single-detection study evaluates the
specificity of the detection antibody. This was conducted
by testing the individual detection antibody in the presence
of multiplexed antigens and capture beads (Table 7).
Data analysis was weighted on the second highest standard
concentration point. The results showed that the degree
of cross-reactivity across the entire panel was below 2%.
Linearity of dilution was assessed by spiking a known
quantity of recombinant antigens into human serum
and plasma samples with 1:3 serial dilutions. For targets
with high endogenous analytes, serum or plasma samples
(with no spikes) were serially diluted one-fourth with sample
diluent. This was followed by a subsequent dilution in
standard diluent to preserve a 25% serum content.
The observed sample concentrations were plotted as a
function of their expected concentrations within the assay
working range to derive correlation coefficient (R2) values.
The results in both single- and multiplex assays in serum
and plasma are presented in Table 8.
Assay parallelism was investigated by comparing the slope
of a spike concentration-response curve in a human serum
or plasma matrix with that of the standard curve using
4-PL curve fitting (Table 8). The percentage difference for
most targets was less than 25%. The data are presented
for information as the results may vary based on
Assay Validation with Biological Samples
The multiplex assays were further validated with measurements
from serum samples collected from individuals with colon,
breast, and lung cancer plus matching controls. Figure 2
is a heat map representation of the relative levels of each
marker. Statistical differences in measured values were
generated using Bio-Plex Data Pro™ software with the
Mann-Whitney method. P values below 0.05 were considered
Agreement with other Luminex Platforms
The angiogenesis panel was also validated on a
Bio-Plex 3D system to provide users the option of using
alternative Luminex platforms. Overall, the standard curves
generated with these two instruments are comparable
(Figure 3), with all samples recording a <20% difference
in observed concentration (data not shown).
In recent years, academic and pharmaceutical research
communities have adopted the Luminex platform as
mainstream technology in the life science industry.
The newly configured Bio-Plex Pro human cancer biomarker
panel 1 is a magnetic bead-based assay developed to
gain a global view of angiogenesis and other important
cancer-related processes in one simple assay. It offers
improved quantification, rapid interpretation, ease of
execution, and reproducibility.
The validation studies described here demonstrate the
robustness of the panel of 16 assays. Assay precision,
accuracy, specificity, sensitivity, and linearity were
evaluated according to standard industry guidelines on
validation of immunoassays. By measuring multiple markers
simultaneously, the human cancer panel reduces time,
cost, and sample volume compared to more traditional
systems such as ELISA.
- Bridges EM et al. (2011). The angiogenic process as a therapeutic target
in cancer. Biochem Pharmacol 81, 1183–1191.
- Dale E et al. (2008). Second generation multiplex immunoassays.
BioRadiations 125, 16–21.
- Staton CA et al. (2004). Current methods for assaying angiogenesis in vitro
and in vivo. Int J Exp Path 85, 233–248.
- Staton CA et al. (2009). A critical analysis of current in vitro and in vivo
angiogenesis. Int J Exp Path 90, 195–221.
The Bio-Plex suspension array system includes fluorescently labeled
microspheres and instrumentation licensed to Bio-Rad Laboratories, Inc.
by the Luminex Corporation.