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Surface plasmon resonance application in prostate cancer biomarker research

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DOI: 10.1515/chempap-2015-0053

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 Chemical Papers 69 (1) 143–149 (2015)
DOI: 10.1515/chempap-2015-0053

ORIGINAL PAPER

Surface plasmon resonance application in prostate cancer

biomarker research

a
Pavel Damborský, b Narayanan Madaboosi, b Virginia Chu, b,c
João P. Conde,
a
Jaroslav Katrlík*

a Department of Glycobiotechnology, Institute of Chemistry, Slovak Academy of Sciences,

Dúbravská cesta 9, 845 38 Bratislava, Slovakia

b INESC Microsistemas e Nanotechnologias (INESC MN) and IN-Institute of Nanoscience and Nanotechnology,
Rua Alves Redol 9, 1000 029 Lisbon, Portugal

c Department of Bioengineering, Instituto Superior Técnico, 1049-001 Lisbon, Portugal

Received 15 April 2014; Revised 5 August 2014; Accepted 25 August 2014

Prostate cancer (PCa) diagnostics can be effectively addressed using sensor-based approaches.
Proper selection of biomarkers to be included in biosensors for accurate detection becomes the
need of the hour. Such biosensor and biochip technologies enable fast and efficient determination
of proteins and provide a remarkable insight into the changes in the protein structure, such as
aberrant glycosylation, which can increase the performance, sensitivity and specificity of clinic
assays. However, for a thorough comprehension of such complex protein modifications, it is crucial to
understand their biospecific interactions. Surface plasmon resonance (SPR), one of the most rapidly
developing techniques for measuring real-time quantitative binding affinities and kinetics of the
interactions of antigens and antibodies, was chosen as an appropriate tool for this purpose. Herein,
experiments on the interactions of antibodies specific against different epitopes of free and complexed
prostate-specific antigen (PSA), a prominent PCa biomarker, are presented with two main aims: (i)
to continue as lectin glycoprofiling studies and; (ii) to be used in microfluidic immunoassay-based
platforms for point-of-care devices. Various PSA-specific antibodies were covalently immobilized on
the biochip surface via amine coupling, and free or complexed PSA was injected into the dual-flow
channels of the SPR device. Kinetic parameters and affinity constants of these interactions, as well as
cross-reactivities of the used antibodies were determined. The sandwich assay for PSA determination
was developed employing both primary and secondary anti-PSA antibodies. Sensitivity of the assay
was 3.63 nM−1 , the detection limit was 0.27 nM and the SPR biosensor response towards free PSA
was linear up to 25 nM. All these findings are essential for proper design of a selective, sensitive,
and highly reliable biosensor for PCa diagnosis as a lab-on-chip device.

c 2014 Institute of Chemistry, Slovak Academy of Sciences

Keywords: biomarker, prostate-specific antigen, prostate cancer, antibody, surface plasmon reso-
nance, biospecific interaction

Introduction nosis, prostate-specific antigen (PSA) assay is used

Prostate cancer (PCa) represents one of the most
common malignancies in men globally (Gilgunn et al.,
2013). It is also the second leading cause of death
after lung cancer (Madu & Lu, 2010). For its diag-
as a traditional method for early detection of PCa
(Gilgunn et al., 2013). Serum PSA is a glycopro-
tein found in either free, unbound form (free PSA;
fPSA), or bound form (complexed PSA; cPSA) with
plasma proteins, mainly the serine protease inhibitor

*Corresponding author, e-mail: chemjkat@savba.sk

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144 P. Damborský et al./Chemical Papers 69 (1) 143–149 (2015)
α1-antichymotrypsin (ACT) (Prensner et al., 2012).
Total PSA (tPSA) is the sum of fPSA and cPSA.
Even though the PSA biomarker assay is the most
widely used test for PCa screening, the PSA level is
associated with considerable specificity and sensitiv-
ity problems, especially in the diagnostic “grey zone”
in the range of 4–10 ng mL−1 (0.13–0.33 nM) (Goo
& Goodlett et al., 2010). Furthermore, the PSA assay
cannot distinguish between PCa and other prostate
abnormalities like benign prostate hyperplasia (BPH)
and prostatitis (Fukushima et al., 2010; Velonas et
al., 2013). Generally, PSA is produced not only by
malignant cells but also by non-cancerous secretory
cells of the prostate (Isono et al., 2002). Consider-
ing the specificity and sensitivity issues related to
the detection of PSA in PCa screening assays, this
concept has proved to be controversial having several
limitations such as PCa overdiagnosis and overtreat-
ment (Prensner et al., 2012). Nowadays, it is largely
abandoned and the predictive value of PSA itself is
considered to be unsatisfactory. Thus, to increase the
specificity of PSA, different parameters have been pre-
sented, e.g. PSA velocity (change of the PSA level
over a time period), PSA density (ratio of PSA to
the prostate volume), cPSA/fPSA and fPSA/tPSA
ratios, or age-/race-specific reference ranges (Stephan
et al., 2014). Nevertheless, all these PSA based assays
have been only partially successful. In order to find
a reliable diagnostic tool for early detection of PCa,
new approaches, such as monitoring of PSA aber-
rant glycosylation in tumor progression, which rep-
resent a challenge in the development of PCa diagno-
sis have emerged (Gilgunn et al., 2013). It is known
that changes in protein glycosylation play the main
role in various physiological and pathological pro-
cesses (including tumor progress) (Kuzmanov et al.,
2013); moreover, significant differences in the glycosy-
lation patterns between PSA obtained from cancerous
and non-cancerous tissues have been reported (Isono
et al., 2002; Gilgunn et al., 2013).
To fulfill the diagnostic potential of PSA related
to all discussed approaches to develop point-of-care
devices, it is necessary to understand the biospecific
interactions involved. Presently, the most appropriate
method for the analysis of such interactions is the sur-
face plasmon resonance (SPR) technique. Essentially,
an SPR biosensor is a label-free technique based on the
measurements of the interaction of an analyte with an
immobilized ligand. Binding of the free analyte causes
a change in the local refractive index monitored in
real-time. Several studies have shown that the SPR
method is well suited for the study of biospecific inter-
actions as well as for the determination of the kinetics
and affinities of analyte/ligand interactions, including
PSA (both free and complexed PSA) (Haseley et al.,
1999; Katsamba et al., 2006; Cao et al., 2006; Katrlík
et al., 2011; Safina et al., 2011; Jeong et al., 2013). The
study of these interactions is also an important step
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in the development of point-of-care devices enabling
fast, cheap and reliable diagnostics of prostate cancer
based on immunoassays and microfluidic platforms.
In the present study, the immunosensor technique
for the measurement of biospecific interactions of
PSA/anti-PSA antibody (Ab) pairs was evaluated.
Kinetic and affinity parameters of PSA and the dif-
ferent Abs tested were determined. The construction
of an SPR biosensor based on the sandwich assay with
a secondary Ab was performed followed by the evalu-
ation of its analytical characteristics.
Experimental
General
Experiments were performed using a dual chan-
nel Reichert SR7000DC SPR system controlled by
the SPR Autolink System software recording also sen-
sorgrams (AMETEK, Reichert Technologies, USA).
The SPR flow cell was equipped with a research-
grade carboxymethyldextran hydrogel sensor chip
CMD50m (Xantec Bioanalytics, Germany). Amine-
coupling reagents (N-ethyl-N-(3-dimethylaminopro-
pyl) carbodiimide (EDC), N-hydroxysuccinimide
(NHS), ethanolamine hydrochloride, sodium dodecyl
sulfate (SDS) and phosphate buffered saline with
Tween
20 (PBST) were purchased from Sigma–
Aldrich (USA). Mouse IgG anti-PSA monoclonal
Abs; Ab10185, Ab10187, Ab10189 (2 mg mL−1 , in
0.1 mass % sodium azide) were purchased from
Abcam (UK) and directly used without any fur-
ther purification. fPSA and complexed PSA with-
ACT (PSA/ACT) purified from human seminal fluid
were obtained from Fitzgerald Industries International
(USA). General-use chemical reagents were purchased
from Sigma–Aldrich.
Chip preconditioning and ligand immobiliza-
tion
A chip was preconditioned before the immobiliza-
tion by two consecutive pulses of 10 L of 100 mM
HCl, 50 mM NaOH, 0.5 mass % SDS, and water in-
jected at the flow rate of 100 L min−1 according
to Katsamba et al. (2006). Immobilization of Abs to
the carboxymethylated dextran (CMD) matrix on the
CMD50m sensor chip was performed at the flow rate
of 20 L min−1 employing the procedure for amine
coupling recommended by the manufacturer. For anti-
PSA Ab immobilization, the CMD matrix was acti-
vated for 7 min (both working and reference chan-
nels of the SPR flow cell) using a fresh mixture of
0.2 M EDC and freshly prepared 0.05 M sulfo-NHS,
which was followed by an Ab injection for 10 min
(only working channel). The mouse monoclonal anti-
PSA Abs concentration used for the immobilization
was 16 g min−1 in a 10 mM sodium acetate buffer
 P. Damborský et al./Chemical Papers 69 (1) 143–149 (2015)
(pH 5.0). The immobilization procedure was ended by
an injection of 1 M ethanolamine hydrochloride for
10 min (both channels).
Surface plasmon resonance measurements
Analyses were performed at 25 ◦C. PBST was used
as the running buffer at the flow rate of 50 L min−1 .
Kinetic analysis was started by an injection of fPSA or
PSA/ACT dissolved in the running buffer at the con-
centrations of 0.39–100 nM. Injection times for both
fPSA and PSA/ACT were 3.5 min, followed by a dis-
sociation period of another 3.5 min. The chip surfaces
were regenerated by a 1 min pulse of 20 mM HCl. The
SPR response was expressed in arbritrary refractive
index units (RIU) as a difference between the signal
of the working and the reference channels.
Antibody specificity
The mouse IgG anti-PSA monoclonal Abs used
were Ab10185 specific for epitope 5 (both fPSA and
PSA/ACT), Ab10187 specific for epitope 1 (only
fPSA) and Ab10189 specific for epitope 3 of PSA
(both fPSA and PSA/ACT). For the sandwich assay,
after a wash with PBST, the specific Ab was injected
into the channel, allowing association and dissociation
time of 3.5 min each.
Evaluation of kinetic parameters
Data processing and kinetic fitting were performed
using the Scrubber software (version 2.0c; BioLogic
Software Pty, Australia). Data processing eliminates
systematic imperfections (caused by baseline drift,
bubbles, refractive index changes, injection noise, etc.)
and results in reliable data for kinetic analysis and ki-
netic fitting, respectively. The responses were fit to a
simple 1 : 1 equilibrium association model by Eq. (1):


Ckon Rmax 1 − e−(Ckon +koff )t
Rt = (1)
Ckon + koff
where Rt represents the concentration of complex
antigen–Ab on the film surface, C is the concentration
of the protein and t represents the time, kon is associ-
ation rate constant, koff is dissociation rate constant
and Rmax is the maximum binding signal. Dissocia-
tion of antigen–Ab complex was fitted by Eq. (2) (Hu
et al., 2007; Mani et al., 2012):
Rt = Rmax e(−koff t) (2)
The Scrubber software employs these equations to
find the minimum sum of squares giving the best val-
ues of kon , koff and Rmax . The equilibrium–binding
constant (KD ) was determined using Eq. (3):
koff
KD = (3)
kon
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145
Fig. 1. Sensorgram of the Ab10187 molecules immobilization
on the chip surface after preconditioning: EDN/NHS
surface activation (1), Ab immobilization procedure
(2), blocking with ethanolamine (3). Lines represent
signals of the working channel (dashed blue), reference
channel (dotted red) and the difference between the two
channels (solid rose). The arrow shows the Ab immo-
bilization level.
On basis of Eqs. (1)–(3), the values of kon , koff and
KD were determined by fitting the sensorgrams of a
set of PSA concentrations.
Results and discussion
Immobilization
All Abs were immobilized according to the same
protocol. Ab incubation time of 10 min resulted in
an immobilization level of 3563 RIU for Ab10189,
2730 RIU for Ab10187, and 4075 RIU for Ab10185.
The estimated amount of immobilized Abs was 3.56
ng mm−2 for Ab10189, 2.73 ng mm−2 for Ab10187,
and 4.08 ng mm−2 for Ab10185, based on the assump-
tion that the immobilization level of 1000 RIU corre-
sponds to the immobilization of approximately 1 ng of
protein per mm2 . A sensorgram of the immobilization
step is shown in Fig. 1.
Interaction of PSA with antibodies
Fig. 2 shows a schematic representation of the
experimental set-up of the performed measurements.
First, an interaction of immobilized Abs with fPSA
was tested (Fig. 2a). Then, the sandwich assay was
applied using primary and secondary Abs selected ac-
cording to the determined interaction characteristics,
association and dissociation constants, and the fPSA
response level (Fig. 2b). Details of the sandwich assay
are discussed later in the paper.
Three commercially available PSA-specific Ab
(namely Ab10189, Ab10187 and Ab10185) were used
to study PSA – anti-PSA interactions. From the pre-
liminary experiments resulted that Ab10189 has very
low affinity towards fPSA (data not shown) and the
146 P. Damborský et al./Chemical Papers 69 (1) 143–149 (2015)

Table 1. Kinetic parameters of fPSA and PSA/ACT interactions with individual PSA-antibodies

Antigen Antibody 105 · kon /(M−1 s−1 ) 10−3 · koff /s−1 10−8 · KD /M

fPSA Ab10189 N.A. N.A. N.A.

fPSA Ab10187 0.36 0.77 2.17
fPSA Ab10185 2.54 4.45 1.75
PSA/ACT Ab10185 2.45 1.78 0.73

N.A. – Not applicable.

Fig. 2. Experimental set-up, immobilized Ab on a CMD sensor chip with bound fPSA or PSA/ACT (a), sandwich configuration
with primary Ab and secondary Ab (b).

signal was below the detection limit, which indicates

unsuitable spatial arrangement of Ab10189 after the
immobilization providing no kinetic data or affinities
of this Ab molecule. The measured responses of fPSA
towards both Ab10187 and Ab10185 were significantly
higher than that towards Ab10189. Reproducibility
of the measurements of two consecutive injections of
25 nM fPSA is depicted in Fig. 3. For fPSA and
Ab10189, the obtained kon = 3.55 × 104 M−1 s−1 , koff
= 7.70 × 10−4 s−1 and KD = 2.17 × 10−8 M. The in-
teraction of fPSA towards Ab10185 was stronger with
kon = 2.54 × 105 M−1 s−1 , koff = 4.45 × 10−3 s−1 and
KD = 1.75 × 10−8 M (Fig. 4). Although the dissocia-
tion constants for both Abs were nearly identical, ki-
netic parameters of binding revealed a ten-fold higher
kon and a ten-fold lower koff for Ab10185 (Table 1). Fig. 3. Sensorgram of two consecutive injections of 25 nM
Hence, for the following experiments with PSA/ACT, fPSA on the sensor chip with immobilized Ab10185 in-
cluding all measurement steps: injection of PSA (1),
the choice of the optimal binder should be based on
association of PSA to Ab (2), dissociation of PSA from
the kon and koff values. Ab (3) and regeneration (4).
Experiments on the interaction of PSA/ACT
only towards Ab10185 were performed (Fig. 5) since
Ab10189 yielded very low signals and Ab10187 is
not specific against PSA/ACT. The values kon = scribed experiments differ in the methodology, type
2.45 × 105 M−1 s−1 , koff = 1.78 × 10−3 s−1 and and number of antibodies as well as in biochip de-
KD = 7.29 × 10−9 M were obtained. Although the sign. In general, kinetics and affinities determined us-
response level of PSA/ACT was lower compared to ing methods in which one of the binding pair is immo-
that of fPSA, the determined value of KD suggests a bilized on a solid support should be seen as apparent
ten-fold higher affinity of PSA/ACT towards Ab10185 since they depend not only on the chemical nature of
compared to that of fPSA. the observed molecules but they are also affected by
The kinetics and affinities of anti-PSA – PSA inter- the immobilization itself (method, density), used car-
actions obtained (Table 1) agree with those obtained rier, etc. They are thus suitable mainly for the com-
using SPR presented in literature (Table 2). The de- parison of the interactions of one immobilized ligand

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 P. Damborský et al./Chemical Papers 69 (1) 143–149 (2015) 147
Table 2. Overview of reported kinetics and affinities of anti-PSA–PSA interactions determined by SPR

PSA anti-PSA 105 · kon /(M−1 s−1 ) 10−5 · koff /s−1 10−10 · KD /M Reference

fPSA commercial 0.25–0.48 4.05–6.40 9.40–0.17 Katsamba et al. (2006)

fPSA commercial 0.73–4.58 2.05–92.90 1.56–0.01 Nagasaki et al. (1999)
fPSA prepared N.A. N.A. 55.00–0.01 Black et al. (1999)
PSA/ACT commercial 0.64–2.50 2.88–33.40 1.19–51.90 Nagasaki et al. (1999)
PSA commercial 1.90 63.00 33.00 Karlsson et al. (2006)

N.A. – Not applicable.

Fig. 4. Fitted curves (dashed) of experimental sensorgrams (solid) (a, b) with corresponding residuals (c, d) showing the interaction
of Ab10185 (a) and Ab10187 (b) immobilized on the sensor chip with various concentrations of fPSA: 100 nM, 50 nM,
25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM and 0.39 nM, respectively (a); 100 nM, 50 nM, 25 nM, 12.5 nM,
6.25 nM and 3.13 nM, respectively (b).

with various molecules. When comparing the results
of different studies, the results need to be interpreted
with respect to the above-mentioned factors accord-
ingly.
Sandwich assay
A typical sandwich assay configuration was used to
demonstrate the biospecific interactions of PSA and
anti-PSA antibodies. The choice of primary Ab was
based on the koff values; those representing the slow-
est dissociation rates were used. Thus, for the follow-
ing experiment, Ab10187 was chosen as a more appro-
priate candidate for this purpose. Additionally, the Ab
providing higher signal is more useful as the secondary
Ab in the sandwich assay design (Fig. 2) because the
intensity of the evanescent waves and thus of the SPR
signal decreases exponentially with the distance from
the sensor surface. Therefore, Ab10185 was selected
as the secondary Ab for the sandwich assay.
In a sandwich assay, the primary and the secondary
Abs must not react with each other. Hence, to en-
sure that the Ab components within an assay do not
contribute to the signal measured, the cross-reactivity
control was done considering all possible Abs pairs.
The results suggest that the individual PSA Abs do
not cross-react with each other (signal obtained with
such pairs is within the noise level and hence it is
negligible) and only the biospecific PSA–Ab interac-
tions in the assay contribute to the signal. A sandwich
configuration consists of two recognition steps. In the
first step, an Ab (Ab10187 specific for fPSA, epitope

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148 P. Damborský et al./Chemical Papers 69 (1) 143–149 (2015)
Fig. 5. Fitted curves (dashed) of experimental sensorgrams
(solid) (a) with corresponding residuals (b) showing
the interaction of Ab10185 immobilized on the sensor
chip with various concentrations of PSA/ACT: 100 nM,
50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.13 nM and 1.56 nM,
respectively.
1) immobilized on a sensor chip surface was allowed
to bind with the injected fPSA. In the second step,
a secondary Ab (Ab10185 specific for both fPSA and
PSA/ACT, epitope 5) targeting yet another epitope
of the antigen (fPSA) is added to bind with the pre-
viously captured fPSA. The two separate recognition
steps lead to enhanced sensitivity and specificity. The
signal was measured twice: at the end of the associa-
tion phase after PSA injection, and at the end of the
association phase after secondary Ab injection. Fig. 6
shows calibration curves of the primary SPR response
(without secondary Ab) and of the sandwich response
(with secondary Ab). The linear regresion equation
for the primary response of fPSA towards Ab10187
immobilized on the sensor surface was calculated as:
y = 1.1892x + 0.1095 (4)
with the linear regression coefficient (R2 ) of 0.9974,
where y and x are the response in RIU and the an-
alyte concentration in nM, respectively. The limit of
detection (LOD) was determined to be 1.30 nM. Sim-
ilarly, the linear regression equation for the sandwich
response was:
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Fig. 6. Calibration curves of primary SPR response (without
secondary Ab) ( ) and sandwich assay (with secondary
•
Ab) ( ). Ab10187 was immobilized on the sensor chip,
fPSA was injected over the surface, and followed by an
injection of the secondary antibody Ab10185.
y = 3.6309x − 0.4258 (5)
(R2 = 0.9996); LOD derived from this equation was
0.27 nM. The slopes of the equations show that the
sensitivity was improved by a factor of 3.05 by the
sandwich enhancement strategy. The response to PSA
was in both cases linear at least up to 25 nM of fPSA.
Hence, it is useful in cases where signal amplifica-
tion is necessary and thus applicable in the enhance-
ment of assays where molecules with multiple epitopes
are employed. The sandwich assay enables specific de-
tection even of low levels of antigens that can be clin-
ically, environmentally, and industrially relevant. The
secondary Ab can be also modified by nanoparticles
for further significant enhancement of the sensitivity
of SPR detection. Uludag and Tothill, (2012) have pre-
sented a sandwich SPR assay using Ab-modified gold
nanoparticles for the determination of tPSA in real
samples. The response to tPSA (prepared as a mass
ratio 1 : 1 mixture of PSA and PSA–ACT) was linear
up to 100 ng mL−1 and the detection limit was 0.5
ng mL−1 of tPSA in the buffer.
Conclusions
The present study shows that the SPR technique
can be effectively used to analyze and quantify biospe-
cific interactions. This approach can be exploited to
study the affinity and interaction mechanism of PSA
and the specific monoclonal Abs involved. It can also
be used to improve the Ab-based diagnostics assays,
thus extending its applications to clinically relevant
limits. The obtained results represent the initial step
of further glycoprofiling of PSA using lectins as well
as of the development of a microfluidic detection plat-
form based on immunoassays thus improving PCa di-
agnostics.
 P. Damborský et al./Chemical Papers 69 (1) 143–149 (2015) 149

Acknowledgements. This work was funded by the European Katrlík, J., Škrabana, R., Mislovičová, D., & Gemeiner, P.
Commission FP7 Programme through the Marie Curie Initial (2011). Binding of D-mannose-containing glycoproteins to
Training Network PROSENSE (grant no. 317420, 2012-2016). D-mannose-specific lectins studied by surface plasmon reso-
This work has been supported by grants VEGA 2/0162/14 and nance. Colloids and Surfaces A: Physicochemical and Engi-
APVV-0290-10 and this publication is the result of the project neering Aspects, 382, 198–202. DOI: 10.1016/j.colsurfa.2011.
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– Stage II supported by the Research & Development Opera- Thornton, K., Zhu, M., Bos, T. V., Forte, C., Friend, D.,
tional Programme funded by the ERDF. INESC MN acknowl- Laird-Offringa, I., Tavares, G., Whatley, J., Shi, E., Widom,
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