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Introduction
Tumors, especially the malignant tumor, are still one of
the leading causes of death in the world. Early screening
and detection of suspected cancer patients is the key to
improving the therapeutic effect. However, early diagnosis
is difficult because clinical presentations are highly variable
and signs are often subtle and common to a variety of
conditions. A tumor marker is a substance sometimes found
in an increased amount in the blood, other body fluids, or
tissues and may mean that a certain type of cancer is in the
body. The screening test by tumor marker has been proven
to be an effective method for detecting cancer in
asymptomatic individuals[1,2]. An ideal tumor marker would be of 100%
sensitivity and specificity, reflect the change in tumor burden,
allow for therapeutic intervention, and predict prognosis and
recurrence. Unfortunately, the tumor markers available
nowadays can not meet all these characteristics. For example,
only 50%_70% of hepatocellular carcinoma patients showed
a significant enhancement in the amount of
alpha-fetoprotein (AFP) in sera. However, the use of panels with 2 or more
approved markers can contribute to the improved
sensitivity and diagnostic efficiency. Therefore, an analysis of the
complete set of tumor markers is often of more value than an
analysis of a single isolated marker.
Recent advances concerning the applications for the
simultaneous detection of various molecules have resulted in
different microsphere-based flow cytometric assays (MFCA).
The MFCA developed by Luminex (Austin, TX, USA) involves the covalent coupling of a capture antibody or target
molecule on polystyrene microspheres (5.6 µm). The
microspheres are internally dyed with red and orange
fluoro-phores. By varying the ratio of the 2 fluorophores, up to 100
different bead sets can be distinguished and each bead set
can be coupled to a different biological probe. A reporter
molecule labeled with a fluorescent marker (such as
phycoerythrin [PE] or Alexa-532) binds to the analyte captured on
the beads. The bound microspheres are passed though a
flow cytometer specially designed to identify the different
microsphere sets. Quantification is based on the intensity
of the fluorescent reporter signal. Previous studies have
used this type of technology for multiplexed assays of
cytokines, antibodies, hormones, nucleic acids, viruses, and
other biomolecules[3,4]. However, at present, either the
specific antibody pairs or the availability of predefined kits
limits these assays. In addition, earlier investigations did not
report the reproducibility of the protein-microsphere
coupling procedure, which is crucial for long-term, particle-based
flow cytometric assays.
To overcome these limitations, in the present study we
chose to develop and validate our own MFCA to quantify
tumor markers as a means to simultaneously measure the
levels of human AFP, carcinoembryonic antigen (CEA),
cancer antigen (CA)19-9, CA24-2, and CA72-4. We chose these
5 common abdominal tumor markers in order to screen and
detect suspected cancer patients. We compared the
performance of MFCA with that of a conventional ELISA system.
Specifically, we determined whether the imprecision in
reproducing the coupling of capture antibodies to beads at
different times affected the results obtained in assays of
human tumor markers. We also compared the upper and
lower limits for the detection of human tumor markers using
MFCA and ELISA. Finally, we compared the reproducibility
of MFCA for measuring tumor markers in human serum
samples. We showed that MFCA is a reliable, fast, and
reproducible technique with a sensitivity that is comparable
to that of conventional ELISA.
Materials and methods
Serum sample preparation Blood samples were collected
from 10 patients with abdominal tumors, including 4 patients
with hepatocellular carcinoma, 4 patients with colon cancer,
1 patient with pancreatic carcinoma, and 1 patient with
cholangiocarcinoma at the initial diagnosis of the diseases,
as well as 4 healthy, adult control donors. All participants of
this study signed an informed consent approved by the
Institutional Review Board. Blood samples (5 mL) were
collected in glass tubes without additives and allowed to clot at
room temperature for 60 min. The serum was separated by
centrifugation at 3000×g for 30 min. Aliquots of serum
(150 µL) were taken and stored at -80 °C until ready for
use. The time from collection to frozen storage was no
more than 3 h. The samples were collected and contained
no identifying features that would make it possible for the
investigators participating in the study to identify the
patients.
Reagents Carboxylated polystyrene microspheres
numbers 101, 103, 105, 107, and 109, each with a distinct emitting
fluorescence pattern, were purchased from Luminex (USA).
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) were
provided by Pierce (Rockford, IL, USA).
2-(N-Morpholino))ethanesulfonic acid (MES), streptavidin-R-PE (SA-PE),
streptavidin-Alexa-532, and streptavidin-horseradish
peroxidase were from Sigma (St Louis, MO, USA). PE-conjugated
goat antimouse immunoglobulin G (IgG) was supplied by
Santa Cruz Biotechnology (Santa Cruz, CA, USA). The
DSB-X biotin protein labeling kit was provided by Molecular
Probes (Eugene, OR, USA).
Human AFP, CEA, CA19-9, CA24-2, and CA72-4 mouse
monoclonal antibody pairs, directed against different
non-competing epitopes of their respective targets, and standard
recombinant proteins were purchased from Biodesign (Saco,
ME,USA). First, azide, tris, glycine, or other
nitrogen-containing compounds were removed by dialysis overnight in
phosphate-buffered saline (PBS) using Slide-A-Lyzer
cassettes (Pierce, USA) since these substances can interfere
with the coupling reaction. All recombinant proteins were
reconstituted in PBS (pH 7.4) containing 1% bovine serum
albumin (BSA) to a concentration of 10 μg/mL. All
proteins were aliquoted and stored at -20 °C.
Covalent coupling of capture antibodies to fluorescent
microspheres Covalent coupling was performed by
following the protocols recommended by Luminex with some
modifications[5,6]. In short, after being washed twice with 200 µL
activation buffer (0.1 mol/L
NaH2PO4 [pH 6.2]), spectrally
differentiable carboxylated microspheres
(2.5×106) were pelleted (10
000×g for 5 min) in 1.5 mL Eppendorf tubes in a
microcen-trifuge (Eppendorf, Hamburg, Germany).
The microspheres were resuspended by sonication (30 s at 1120 W, 50_60 Hz)
and gentle vortexing (VWR International,
West Chester, PA, USA) in 80 µL activation buffer to
which 10 µL of each solution of sulfo-NHS and EDC (both at 50 mg/mL), prepared in
ddH2O immediately before use, was sequentially added to
stabilize the reaction and activate the microspheres. The
suspension was allowed to incubate for 20 min at room
temperature and then resuspended in 500 µL coupling buffer
[0.05 mol/L MES (pH 5.0)] containing 125 µg capture
antibody. The mixture was incubated for 2 h in the dark with
continuous shaking. The 250 µg/mL concentration of the
antibody was used for coupling because our
preliminary experiments indicated that this coupling concentration yielded
high median fluorescent intensities (MFI) and good
inhibition (data not shown). The coupled microspheres were then
incubated in 500 µL blocking buffer [PBS-TBN: PBS
containing 0.1% BSA, 0.02% Tween-20, and 0.05%
NaN3 [pH 7.4)] for 30 min to block the uncoupled sites. An aliquot of the
microspheres was diluted at 1:5 in PBS-TBN to count 2 of the
9 large squares on the grid on the hemacytometer (Bright
Line, VWR International, West Chester, PA, USA). The
concentration was calculated as follow: Bead count/2
(No squares)×5 (dilution
factor)×10 000=microspheres/mL. Finally, the concentration of each bead set was adjusted to
1×106/mL with PBS-TBN and stored at 4 °C in a light-safe
container. For the studies of the reproducibility of coupling
and its effect on subsequent assays of tumor markers, 5
independent coupling reactions were performed over a
period of 1 week.
Testing the efficiency and density of antibody coupling
to the microspheres After prewetting the 1.5 mL Eppendorf
tube with assay buffer (PBS containing 1% BSA [pH 7.4]), 50
µL of the antibody-coupled microsphere sets (at a final
concentration of 100 microspheres of each set/µL) and 50 µL
PE-conjugated goat antimouse IgG detection antibody (2-fold
serial dilution from 4 to 0.0625 µg/mL) were added into the
tube. After incubation for 30 min and washing twice with
PBS-1% BSA, the microspheres were measured and analyzed
with the Luminex 100 system. The capture antibody density
was measured by computing the ratio of the MFI of the
coupled and uncoupled beads that reacted with the
PE-conjugated goat antimouse IgG secondary antibody.
Detection of non-specific cross-reactivity in multiplexed
MFCA The biotin labeling of the detection antibody was
performed by following the commercial kit specifications.
To detect whether non-specific cross-reactivity between
components during the assay occurred, and consequently, to
monitor false positive findings, a full microsphere mixture
with the biotinylated detection antibodies for each
microsphere were incubated in the presence of a single
tumor marker standard at 64 µg/mL (AFP and CEA) or 64 U/mL
(CA19-9, CA24-2, and CA72-4). After incubation for 30 min
and washing twice with PBS-1% BSA, the microspheres were
measured and analyzed with the Luminex 100 system.
Multiplex tumor marker assays The establishment of
the standard curve of MFCA was prepared with 4-fold serial
dilution steps (from 64 to 0.001 µg/mL for AFP and CEA or
from 64 to 0.001 U/mL for CA19-9, CA24-2, and CA72-4) of
recombinant protein standards of tumor markers. Each
standard was tested in a single bead assay to determine the
optimal concentration of the detection antibody. Next, the
microspheres were multiplexed and optimized for the
incubation times and reporter signal. As a reporter signal,
both streptavidin-Alexa-532 and SA-PE were tested in
different concentrations. The samples were measured twice
and blank values were subtracted from all
readings. All assays were carried out directly in a 1.5 mL Eppendorf tube
at room temperature and protected from light. A mixture
containing 5000 microspheres/set was incubated together
with a cocktail of biotinylated antibodies and a standard,
sample, or blank of the indicated concentration in a final
volume of 100 µL for 30 min under continuous shaking.
The microspheres were then washed twice with PBS-1%
BSA in order to remove any unbound antibodies. After 30
min of incubation with SA-PE and washing twice with
PBS-1% BSA again, the microspheres were measured in a final
volume of 100 µL and counted 50 µL and total 50 of each set
of microspheres to obtain the MFI. The analyzer contains 2
solid state lasers: a classification laser (635 nm, 10 mW
maximum) excites the fluorochromes impregnated within the
microsphere, and a reporter laser (532 nm, 100 mW maximum)
excites fluorescent molecules bound to the microsphere
surface. The classification emission spectrum does not
overlap with the reporter emission signal. Therefore,
compensation is not necessary as with a conventional flow cytometer.
Tumor markers ELISA ELISA was performed by
coating 96-well polystyrene plates (Corning, Corning, NY, USA)
with a specific monoclonal antibody. Standards and samples
were added to the appropriate wells after the plates were
blocked with non-fat, dry milk, and the plates were
incubated overnight at 4 °C. A specific biotinylated antibody
was then added to all the wells after they were washed with
PBS, and they were incubated for 1 h at room temperature.
The plates were washed with PBS again and incubated for
another 30 min with horseradish peroxidase-conjugated
streptavidin. After the removal of the non-bound
horseradish peroxidase conjugate by washing, tetramethylbenzidine
dihydrochloride substrate reagent solution (ICN Biomedicals,
Aurora, OH, USA) was added to the wells. The reaction was
stopped with 25 µL/well of 2 mol/L
H2SO4. The plates were read at 450 nm with a microplate spectrophotometer
(Molecular Devices, Menlo Park, CA, USA) and the results
were analyzed with associated software.
Results
Optimization of reagents and procedures Specific
antibody pairs were used to develop and validate this multiple
human tumor marker assay. The critical parameters of this
assay are the choice of primary antibodies, the
concentration of the biotinylated detection antibodies, the amount
and choice of conjugated fluorochromes, and the
incubation times of the samples and the conjugated fluorochromes.
Several commercial antibody pairs suitable for use in ELISA
might not perform well in our multiplex system; either the
coupling to the microspheres would be ineffective or the
beads would fail to report a signal from the recombinant
standards. The test for the coupling efficiency (such as
AFP) showed that the highest coupling readings were
approximately 15 000 MFI, while backgrounds were lower than
100 MFI, which proved that antibody coupling to the
microspheres was successful (Figure 1).
The biotin-conjugated detection antibodies were
optimized in a single bead assay before they were added to the
multiplex profile. The amount of the conjugated antibody
used varied per tumor marker and the optimal
concentrations of detection antibody for this assay ranged between
1.0 and 5.0 µg/mL. As a reporter signal, both
streptavidin-Alexa-532 and SA-PE were tested in different concentrations
ranging from 0.1 to 1.0 µg/mL for Alexa-532 and 0.1 to 5.0
µg/mL for SA-PE in the present study. Alexa-532 turned out
to be less suitable for several of our antibodies because we
were unable to pick up a signal at a different concentration
of fluorochromes, whereas SA-PE resulted in a signal.
Therefore, we used SA-PE for further experiments and the
optimal concentration was 4 µg/mL. Prolonging the
incubation times for either the sample or SA-PE did not improve our
ability to detect any tumor markers. Overall MFI signals
increased with longer incubation times, but sensitivity was
lost due to a proportionally higher increase in the background
signal.
Variability of antibody-bead coupling and its effect on
assays For long-term studies, it is crucial that the process of
coupling the capture antibody to beads can be reproduced
so that bead sets coupled independently will give
consistent assay results. To address this concern, we tested how
the density of capture antibodies on the beads varied during
repeated coupling procedures. We observed that minor
variations in the coupling procedures could markedly affect the
density of coupled capture antibodies. Table 1 indicates the
variation of the coupled antibody density of the beads after
5 independent coupling reactions. The coefficient of
variation for the density of the independently coupled bead sets
ranged from 11.2% (AFP) to 26.1% (CEA). In subsequent
studies, we found that the optimal concentrations of the
capture antibodies for coupling were generally those that
resulted in the greatest density of antibodies coupled to
beads. Beads with high coupling densities tended to give
more sensitive assays than beads with low coupling
densities (Figure 2).
Identification of cross-reactivity As shown in Table 2,
although the background varied with each microsphere, none
of the specific antibody sets gave readings above
background with non-relevant tumor marker standards,
indicating that there was no detectable cross-reactivity. Positive
readings were found for the microspheres labeled with the
specific capture antibody.
Range of detection for standard curves The standard
curves for all 5 tumor markers are represented in Figure 3.
We compared the lower, upper, and total working detection
ranges for ELISA and MFCA single and multiplexed assays
of these 5 tumor marker standards. The approximate upper
and lower limits of detection were conservatively estimated
by comparing the largely linear regions of the standard curves
in which fluorescence and absorbance were plotted linearly
and concentration logarithmically. We excluded the
uppermost region of the multiplexed MFCA where the slopes
differed markedly from those of the individual assays. As shown
in Table 3, ELISA had lower limits of detection compared to
MFCA. For each assay, ELISA was more sensitive by a
factor of 4_16. Conversely, MFCA had a higher detection
limit by a factor of 4_64. The total dynamic ranges were
greater for MFCA than ELISA. In addition, we did not find
that multiplexed MFCA were less sensitive than the
individual assays.
Reproducibility of MFCA in serum samples
The results of MFCA were compared with those of ELISA for serum
samples from healthy individuals and tumor patients with an
unknown concentration of each tumor marker, which would
be widely disparate at the linear part of the standard curves
of the multiplex assay. Correlations between both techniques
were determined by measuring the levels of all tumor
markers with the MFCA system and with conventional ELISA.
All the samples measured with the MFCA system were
assayed twice and at different time points so that the
intra-assay variance could be determined.
The concentrations of the samples measured with ELISA
concurred with the concentrations that were determined with
the MFCA system (for example, AFP in Figure 4). High
correlation coefficients (r2), ranging from 0.927 to 0.985, were
found for all the tumor markers. Slopes varied between 0.86
and 1.03, with a mean slope of 0.95. Intra-assay variability,
expressed as a coefficient of variation, was calculated based
on the average for 14 serum samples and measured twice in
the multiplex assay repeated at 2 different time points. The
intra-assay variability within the replicates of the samples
was comparable to that of ELISA (<10%) with an average
coefficient of variation of 8.92%. Inter-assay variability was
evaluated by testing quadruplicates of 1 standard (1 µg/mL
for AFP and CEA or 1 U/mL for CA19-9, CA24-2, and
CA72-4) that was positioned at the upper linear part of the standard
curve of all the tumor markers in a multiplex assay at 4
different time points. The variabilities of these samples were
between 10.4% and 18.6%, with an average of 15.06%
(Table 4).
Discussion
Tumor markers are soluble proteins that are secreted by
tumor cells or other normal cells stimulated by the tumor
microenvironment and can reflect the existence of a type of
tumor in the body before the presentations of obvious
symptoms. It is at this early stage that most cancer patients
will have the greatest chance of cure. Immuno-assays, such
as ELISA, can provide relatively sensitive, specific, and
precise quantitation of tumor markers, but ELISA can detect
only one analyte in a single test, thus, when applied to the
expanding number of tumor markers that are involved in
defining a particular system, they become expensive and time
consuming to perform. In addition, ELISA has more
limitations, like the need for a large sample volume, the
narrow dynamic range, and complicated dilution
procedures[7,8]. A protein microarray can analyze thousands of proteins
simultaneously, but it can not quantify
accurately[9]. As we know, a test of tumor markers is not simply positive or
negative and it is of great clinical significance when exceeding a
given threshold. Continued technological advances are
expected to provide not only new analyte markers, but also
new technologies to detect them. Here we reported the
establishment of the MFCA system for the detection of
multiple human tumor markers and the results showed that our
multiplexed assay was comparable in sensitivity, accuracy,
and reproducibility to the "gold standard" which was the
ELISA.
The basic principle behind this technology involves
microspheres being labeled with a distinguishable
fluoro-phore that allows it to be assigned or gated to a particular
region by the scanner. Capturing antibodies specific for the
protein of interest is covalently linked to beads of a unique
fluorescent region. The combination of different beads
allows the user to simultaneously measure various proteins
in the flow cytometer. From the immunological point of view,
the limitation of this technique is the availability of matched
antibody pairs that do not cross-react with other reagents.
Numerous matched pairs of high-affinity and
high-specificity antibodies have since been developed and commercialized,
which encourage us to evaluate a multiplexed format of
tumor markers. We have observed that minor alterations of the
coupling process may significantly affect the density of
coupled antibodies. Subsequent assays using bead sets
with a low density of coupled antibodies tended to be less
sensitive and reliable than that with relatively high density.
Thus, it is important to check whether coupled microspheres
have similar densities of capture reagents if repeating the
coupling procedures and to optimize the concentration of
antibodies coupled to beads.
In the present study, we have demonstrated that MFCA
has a clear advantage over conventional ELISA, including
the ability to detect large numbers of analytes
simult-aneously, therefore providing a powerful tool for profiling
multiple tumor markers. Theoretically, this assay can
detect up to 100 analytes in a single sample and thus more
novel markers are readily welcome to be added in this open
system. Consequently, the sample volume required to test a
single marker by ELISA would be sufficient to detect all
analytes by MFCA[10,11]. Obviously, this is a major
advantage when working with a small sample. Furthermore, the
MFCA took 2.5 h not including coupling process nor flow
cytometric analysis. In contrast, ELISA took 5 h nor
including overnight capture antibody incubation and the
spectrophotometric analysis. Alternatively, some commercial kits
containing bead sets already coupled to capture antibodies
are available, and the variety of available kits continues to
expand as this new technology grows. Such kits will
eliminate the time-consuming coupling process and simplify the
detection steps by using automated XY platform (XYP) plate
handler.
In addition, MFCA is sensitive and accurate since this
multiplex assay has been set up as a sandwich immunoassay,
which will enhance specificity and reduce the risk of
cross-recognition with other proteins. Moreover, each
fluorescence signal is the mean of 50 measurements of a single
microsphere, and each microsphere represents an assay by
itself, whereas with ELISA, the samples are usually tested
merely in duplicate or triplicate. Although higher numbers
of events are commonly used in MFCA, using as few as 50
events will not affect the precision of the assay. Within a
liquid phase, the chance of the interaction of proteins with
their complementary antibodies will increase, which will lead
to less non-specific binding and thus to lower background
signals[12_14]. Unlike Carson et
al[ 15], we did not find that multiplexed MFCA were less sensitive than the individual
assays as evidenced by Table 3. However, we tested only 5
not 15 analytes simultaneously in this study, thus, our
multiplexed assays were less likely to be adversely affected by
antibody cross-reactivity and interference.
Moreover, each standard curve can be adjusted to a
biological range in which a sample can be expected depending
on the origin of the specimen. With this wide dynamic range
of standard curves, samples do not have to be diluted or
concentrated[16,17], but it is crucial to confirm that these
parameters are achieved with high-affinity antibodies that
define the lower end of sensitivity of the assay and the rapid
reaction kinetics. Multiplexed assay performance is mainly
dependent on the quality of the antibodies. Therefore,
potential antibodies should be screened initially by comparing
the slopes of single and multiplexed standard curves to
detect possible cross-reactivity problems with the multiplexed
reagents.
We have shown that MFCA can be used to generate
quantitative biomarker data for serum samples, which
appear to be more reproducible than ELISA. MFCA is less
error prone because of its more direct fluorescence
detection system as opposed to the more indirect detection method
of ELISA, which is based on enzyme amplification. However,
this multiplex assay involves mixing a large number of
different antibodies in a single reaction. These antibodies could
bind non-specifically to any other component within the assay
and serve as antigens for other immunoglobulins, causing
false positive values or blocking the readout. Thus, when
sera are used as the matrix, the matrix has to be carefully
monitored for blocking substances like heterophilic
antibodies. In conclusion, MFCA provides a more rapid and
less expensive method that has a 3_4 logarithmic range of
sensitivity compared with 1_2 logs for ELISA, as well as the
specificity and reproducibility expected from the
latter[18]. Therefore, the successful establishment of the MFCA
system for multiple human tumor markers provides the
foundation for the further study of clinical applications.
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