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Biosensors and Bioelectronics 23 (2008) 15471554
Amperometric immunosensor for diagnosis of BLV infection
Bogumila Kurtinaitiene
a,d
, Dovile Ambrozaite
d
, Valdas Laurinavicius
a
,
Almira Ramanaviciene
b,c
, Arunas Ramanavicius
b,
a
Institute of Biochemistry, Mokslininku 12, 2600 Vilnius, Lithuania
b
Center of Nanotechnology and Material Science, Department of Analytical and Environmental Chemistry,
Faculty of Chemistry, Vilnius University, Naugarduko 24, 03225 Vilnius 6, Lithuania
c
Laboratory of Immunoanalysis and Nanotechnology, Institute of Immunology of Vilnius University, Moletu pl. 29, 08409 Vilnius 21, Lithuania
d
Vilnius Gediminas Technical University, Sauletekio al. 11, LT-10223 Vilnius 40, Lithuania
Received 24 September 2007; received in revised form 7 January 2008; accepted 9 January 2008
Available online 18 January 2008
Abstract
A new amperometric immunosensor for detection of antibodies against bovine leukemia protein (gp51) was designed. The detection of
antibodyantigen complex formation was based on application of secondary antibodies labeled with horseradish peroxidase (HRP). Ferrocenecar-
boxylic acid (FCA) and N,N,N

,N

-tetramethylbenzidine (TMB) were selected as suitable mediators for this immunosensor. Optimal conditions for
amperometric detection were found. Sensitivity of created system was compared with the results of enzyme-linked immunosorbent assay (ELISA)
and agar gel immunodiffusion (AGID) reaction, and was sufcient for detection of usual anti-gp51 antibody concentration present in the blood
serum of BLV-infected cattle.
2008 Published by Elsevier B.V.
Keywords: Immunosensor; NanoBioTechnology; HRP; Redox mediator; Electrochemical sensor; TMB; FCA; BLV
1. Introduction
Bovine leukemia virus (BLV) still has a high impact in veteri-
nary. Signicant relationships between BLV-seropositivity and
decreased milk production or mastitis have been reported, most
in large herds with high seroprevalence (Pelzer, 1997). BLV
is still common in Egypt, Pakistan, Canada, Cambodia and
other countries (Meas et al., 2000a,b; VanLeeuwen et al., 2001;
Zaghawa et al., 2002). BLV was registered even in some Euro-
pean countries (Ramanaviciene et al., 2004a,b). BLV belongs
to a family of oncogenic retroviruses, which includes HTLV-1,
HTLV-2, simian T cell leukemia virus 1 and 2 and these retro-
viruses share a common genomic and structural organization
(Sagata et al., 1985). Bovine leukemia virus is associated with
enzootic bovine leukemia (EBL), a disease characterized by a
very extended course that often involves persistent lymphocy-
tosis (PL) and culminates in B-cell lymphoma (Burny et al.,
1988). Majority of retrovirus-induced diseases for a long incu-

Corresponding author. Tel.: +370 60032332; fax: +370 52330987.

E-mail address: arunas@imi.lt (A. Ramanavicius).
bational period showno clinical signs. One of the most effective
steps to prevent spread of such infections is fast and effec-
tive strategy for detection. Agar gel immunodiffusion (AGID)
reaction, enzyme-linked immunosorbent assay (ELISA), poly-
merase chain reaction (PCR) and immunoblotting are applied for
the detection of retrovirus specic antibodies or DNA (Grover
and Guillemain, 1992; Simard et al., 2000; Rola and Kuzmak,
2002).
At present, indirect (serological) detection methods are still
dominating in the laboratory diagnosis of BLV infection. The
AGID reaction and ELISA are predominantly used for rou-
tine BLV diagnosis. To diagnose BLV infection at the early
stages polymerase chain reaction is applied. However, the
main drawback of the traditional techniques are the follow-
ing: (i) AGID is limited by extremely long detection time;
(ii) ELISA is faster but requires a lot of manipulations; (iii)
polymerase chain reaction-based techniques have long detec-
tion time limited by electrophoresis in agarose gel and the
necessity to develop the electrophoreograms by highly poi-
sonous ethidium bromide (Murtaugh et al., 1991). AGID
reaction might be replaced by electrochemical detection sys-
tem based on application of synthetic receptors, however
0956-5663/$ see front matter 2008 Published by Elsevier B.V.
doi:10.1016/j.bios.2008.01.014
1548 B. Kurtinaitiene et al. / Biosensors and Bioelectronics 23 (2008) 15471554
such system is not sensitive enough to detect low concen-
trations of BLV proteins that are present in blood serum
only at the early stage of BLV infection (Ramanaviciene and
Ramanavicius, 2004a). Piezoelectric systems might be applied
to simplify ELISA protocol but such systems are externally
sensitive to external physical factors that are inducing drift
in piezoelectric signal (Ramanaviciene et al., 2004b). Surface
plasmon resonance technique can be also applied for label-
free monitoring of antibodyantigen interactions in real-time
(Kausaite et al., 2007). The PCR-based detection of target
DNA can be signicantly simplied if PCR coupled with
DNA-sensors are applied (Ramanaviciene and Ramanavicius,
2004a). Quenching of nonspecically adsorbed uorescence
agents by conducting polymer matrix might be exploited for
increasing sensitivity of sensors devoted for diagnosis of BLV
(Ramanavicius et al., 2007). In construction of all sensors men-
tioned here combination of electrochemistry and biochemistry
(Ramanaviciene and Ramanavicius, 2002) was successfully
exploited.
Construction of amperometric immunosensors based on
registration of enzymatic reaction is a way that allows construc-
tion of sensitive bioanalytical systems. Horseradish peroxidase
(HRP) is one of the mostly used enzymatic labels for the
design of ELISA and amperometric immunosensors (Campas
and Marty, 2007; Salinas et al., 2005). Usually amperomet-
ric immunosensors utilize redox mediators that are involved
into electron transfer from redox active enzyme center towards
the electrode (Ramanavicius et al., 2006). Major problems in
the construction of amperometric immunosensors are the fol-
lowing: the choice of electrode suitable for immobilization of
agents exhibiting afnity towards analyte and selection of suit-
able electrochemicallyactive substrate able totransfer efciently
electrons from HRP.
The aim of this study was to develop an electrochemi-
cal immunosensor model for the amperometric detection of
anti-gp51 antibodies that are present in the blood serum of
BLV-infected cattle.
2. Experimental
2.1. Chemicals
All chemicals were of analytical grade and used as received.
The solutions were prepared by using HPLC grade water
puried in a Purator-B Glas Keramic (Berlin, Germany).
Hydrogen peroxide stock solution of 0.25 M has been used for
preparation of aliquots and concentration of H
2
O
2
was tested
spectrophotometrically before each sequence of measurements.
The BLV protein gp51, real BLV-not-infected bovine blood
serum sample (BLV-not-infected serum) and BLV-infected
bovine blood serumsample (BLV-infected serum) were obtained
from Biok (Kursk, Russia). The BLV-infected serum con-
tained standardized concentration of antibodies against protein
gp51 (anti-gp51). Horseradish peroxidase-labeled secondary
antibodies (HRP-Ab*) for ELISAbovine leucosis serumscreen-
ing test were received from Institute Pourquier (Montpellier,
France).
Horseradish peroxidase activity 560 U/mg, 4-amino-
antipyrine (AAP) and phenol-4-sulfonic acid (PSA) were
received from Fluka (Switzerland), o-phenylene diamine
(OPD) from Merck (Germany). Ferrocenecarboxylic acid
(FCA), trimethyl hydroquinone, N,N,N

,N

-tetramethylben-
zidine (TMB), N-cyclohexyl-(2-morfolinoethyl) N-cyclohexyl-
(2-morfolinoethyl)carbodiimide meto-p-toluensulphonate
(EDC), graphite rod electrodes (99.999% purity) 3 mm in
diameter, were obtained from SigmaAldrich (St. Louis,
Missouri, USA). Biotin hydrazide was received from Pierce
(Rockford, Il, USA).
2.2. Electrochemical setup
All electrochemical experiments were performed by
potentiostatgalvanostat model VoltaLab PGZ 402 Radiome-
ter Analytical (Villeurbanne Cedex, France) in conventional
three-electrode system consisting of working graphite electrode
(bare or modied as described), platinum plate as an auxiliary
electrode and Ag/AgCl (in saturated KCl) as a reference one
(Laurinavicius et al., 1999). The 0.05 M potassium phosphate
buffer solution (PBS), pH 6.0 or 7.0, containing 0.1 M of KCl
was used as default buffer for all experiments.
2.3. Spectrophotometric setup and spectrophotometric
measurements
UVvisible measurements were performed with a spec-
trophotometer Thermo Spectronic UV- 300 (Unicam
Spectronic, Cambridge, UK) using a PVC spectrophotomet-
ric cuvette of 10 mm optical path length and 1 ml of working
volume. Spectrophotometric measurements were carried out
in 0.05 M phosphate buffer with 0.1 M KCl, pH 7.0 at 20

C.
In all spectrophotometric measurement solutions consisting of
0.29 M HRP, and 0.5 mM the corresponding electron donor
were used. To start the enzymatic reaction hydrogen perox-
ide was added to form initial 12.5 mM concentration of this
substrate in a reaction vessel. Then subsequently the reaction
rate and enzymatic activity of HRP were calculated from the
changes in optical absorbtion at
max
for each redox mediator
used.
2.4. Electrode preparation and pretreatment
Graphite electrodes were preparedas follows: rods of spectro-
scopic graphite (SigmaAldrich, USA) were cut; then electrodes
were polished on ne emery paper (Tufback, Durite P1200,
Allar, Sterling Heights, MI, USA) and then polished with alu-
minum paste, following rinsing of the electrode surface with
distilled water and drying at room temperature. Electrodes were
sealed into silicon tubes to dene constant geometric work-
ing area of graphite electrode. This area was of 0.071 cm
2
.
The graphite working electrodes after mechanical pretreatment
(Ramanavicius et al., 2005a) were ultra-sonicated for 10 min in
distilled water, washed with distilled water, air-dried and kept
in the refrigerator at +4

C.
B. Kurtinaitiene et al. / Biosensors and Bioelectronics 23 (2008) 15471554 1549
2.5. Electrode modication procedure and immunoassay
scheme
After pretreatment the carbon electrode was coated with
3 l of BLV protein gp51 dissolved in PBS buffer, pH 7.0
(15 g/ml concentration), and allowed to dry overnight at +4

C.
After that the electrode was rinsed for 30 min with PBS buffer,
pH 7.0, and for 1 h treated with 0.5% BSA to block nonspe-
cic binding of other proteins to the vacant graphite surface.
Then the electrode was rinsed for 30 min with PBS buffer, pH
7.0. Following the last step, 3 l of differently diluted BLV-
infected serum containing standardized physiological amount
of antibodies against gp51 (anti-gp51) was equally deposited
on the electrode surface and incubated at room temperature
for 1 h. Then the electrode was rinsed for 30 min with PBS
containing 0.05%of Tween-20 (PBS-T) to remove all nonspecif-
ically adsorbed proteins. After that the electrode modied with
antigenantibody immune complex (gp51/anti-gp51) was incu-
bated for 1 h in HRP-labeled secondary antibody (HRP-Ab*)
solution. Commercially available 250 times diluted solution
of HRP-labeled secondary antibody against anti-gp51 (HRP-
Ab*) was applied for this purpose. Then again washing step
for 30 min in PBS buffer, pH 7.0, was applied. Efciency of
gp51/anti-gp51/HRP-Ab* complex formation was examined by
HRP-catalyzed enzymatic reaction. Eight types of electrodes
were prepared using eight different gp51 concentrations, at least
three electrodes were modied by the same concentration of
gp51.
2.6. Amperometric detection
The electrochemically active reaction products were
determined amperometrically, recording steady-state cathodic
current at 200 mV vs. Ag/AgCl, in PBS, pH 7.0, contain-
ing 0.5 mM of H
2
O
2
and 0.5 mM of redox mediators TMB
or FCA. The electrochemical cell containing 1 ml of corre-
sponding solution was investigated under stirring by 200 rpm.
Cycling voltammetry was performed by bare graphite electrodes
in 0.05 M phosphate buffer, pH 7.0, containing 0.1 M KCl in the
same reaction vessel without any stirring. The electrochemically
active materials were added consequently in this sequence: (i)
0.5 mM of redox mediator (AAP, FCA, K
4
[Fe(CN)
6
], Pyrogal-
lol, TMB or OPD), (ii) 5 g/ml of HRP, (iii) 0.5 mM hydrogen
peroxide. CVs were registered before and after of each addi-
tion of the above-mentioned materials (i, ii, and iii). Two CVs
scans with potential sweep rate of 50 mV/s were applied and
the second CVs scan was presented in corresponding plot com-
bining four cycling voltammograms: one before addition of the
mentioned materials and the other three after each addition of
electrochemically active materials (depicted in i, ii, and iii).
2.7. Detection of gp51, anti-gp51 and HRP-Ab* interaction
by reference methods
The AGID test and ELISA were performed to test the activ-
ity of immunochemicals used. The AGID test (Kursk biofactory
Biok, Russia) was based on classic Ouchterlony method of
precipitation on agar gel and was performed as it was described
previously (Miller and Van der Maaten, 1976). Briey, the anti-
gens gp51 were placed in the central well and 2, 4, 8, 16, 32,
64, 128 times diluted BLV-infected serum was placed in the
wells around the antigen. Then the antigens and antibodies were
allowed to diffuse freely in the agar gel towards each other.
Their concentration decreased proportionally to the distance
passed fromthe well. After 2448 h precipitation zones that indi-
cated the equivalent concentrations of gp51 and anti-gp51 were
created between the corresponding wells.
Indirect ELISA was performed on commercially available
microplates for detection of anti-gp51 (Institute Pourquier,
France). The BLV-infected sera obtained from Kursk biofac-
tory Biok (Russia) was consequently 2, 4, 8 . . . 256, 512
times diluted with serum of healthy animal, additionally diluted
50 times in ELISA wells coated with viral antigen and then
incubated at +4

C overnight. The antibodies specic to BLV

antigens present in serum formed gp51/anti-gp51 and remained
bound in the well. Following the washing step, secondary anti-
bodies labeled with HRP (HRP-Ab*) were added and incubated
for 30 min to allow interaction of HRP-Ab* with gp51/anti-
gp51 and formation of immune complexes. After the next well
washing step, the enzyme substrate TMB was added into the
wells. Depending on gp51/anti-gp51/HRP-Ab* surface concen-
tration, HRP is catalysing formation of blue compound from
TMB, which is becoming yellow after the blocking step using
0.5 M sulphuric acid. The intensity of colour in each well
was measured at =450 nmwith ELISA-plate spectrophotome-
ter Multiskan MS from Labsystem (Finland). The results
were considered as positive when S/P was greater than 115%
(S/P %=100 [(OD
450
value of the sample OD
450
value of
the negative control)/(OD
450
value of the mean positive con-
trol OD
450
value of the negative control)]).
2.8. Simulation of amperometric immunoassay conditions
In this experiment, bare graphite electrodes were incubated
overnight in0.05 mg/ml of avidindissolvedinPBS, pH6.0. Then
the electrode was thoroughly washed with PBS to remove non-
adsorbed avidin. After washing, 3 l solution containing various
concentrations of the biotin-labeled HRP was deposited on the
avidin-coated electrode surface. Following 30 min incubation,
similar washing procedure was performed again.
Biotin conjugate with HRP was prepared using the follow-
ing protocol: 10 mg of HRP enzyme was dissolved in 1.0 ml of
HEPES buffer, pH 5.5. The 25 l of biotin hydrazide (50 mM
solution in DMSO) and 12.5 l of EDC (100 mM in HEPES,
pH 5.5) were added to the prepared HRP solution and incubated
overnight at 13

C under mild mixing. Then the dialysis against

0.05 M sodium acetate buffer, pH 6.0, was carried out for 24 h.
3. Results and discussion
Our previous studies on detection of BLV infection were
based: (i) on detection of proviral DNA (Ramanaviciene and
Ramanavicius, 2004b); (ii) electrochemical detection of interac-
tion of molecularly imprinted conducting polymer polypyrrole
1550 B. Kurtinaitiene et al. / Biosensors and Bioelectronics 23 (2008) 15471554
Table 1
Activity of HRP detected spectrophotometrically with different electron donors
Electron donor (nm) Mean activity
a
(U/ml) R.S.D. (%)
OPD 510 3.35 2.0
TMB 650 3.24 2.01
AAP 490 2.63 3.0
K
4
[Fe(CN)
6
] 420 0.10 1.5
FCA 450 0.02 1.8
a
The mean value calculated from three measurements.
with BLV antigens (Ramanaviciene et al., 2004a,b) and (iii)
detection of uorescence of secondary antibodies labeled with
HRP (Ramanavicius et al., 2007). HRP-labeled secondary anti-
bodies were used to conrm analyte binding in previously
mentioned case (ii) and as uorescence label in case (iii). To
increase sensitivity and selectivity of bioanalytical system for
BLV detection simultaneous detection of uorescence and elec-
trochemical signals will be a step forward (Willner et al., 2007).
At the current state of our investigations the development of
a bioanalytical system based on amperometric detection was a
major challenge, which is presented in this study.
We have investigated ve potential redox mediators for HRP
(Hou et al., 2007) that in oxidized and/or reduced forms can be
detected by both ways: electrochemically and spectrophotomet-
rically. Activity of HRP detected spectrophotometrically with
different electron donors is presented in Table 1. The highest
activity of HRP was detected if OPD and TMB were applied
as electron donors. These ndings are in line with ndings pre-
sented by (Hou et al., 2007).
3.1. Cyclic voltammetry study of some redox mediators for
HRP
For the designing of a working electrode carbon rod was
selectedas the most suitable substrate fromusual electrode mate-
rials (Pt, Au, carbon rod, glassy carbon) due to: (i) superior
adsorption of proteins including BLV protein gp51 on its sur-
face, (ii) easy electrochemical detection of redox mediators used
in this study, (iii) insignicant interference of amperometric sig-
nal by hydrogen peroxide which was used as substrate for HRP,
in some cases if distance between HRP and carbon electrode
is not too long the direct electron transfer from carbon elec-
trode towards the HRP might be achieved (Andreu et al., 2007;
Ruzgas et al., 1996; Gorton, 1995). Adsorption procedure was
applied for the immobilization of protein gp51, which after incu-
bation in BLV-infected bovine serum formed antigen/antibody
complex (gp51/anti-gp51) that after incubation in HRP-Ab*
containing solution formed antigen/antibody/(secondary anti-
body) HRP-labeled complex (gp51/anti-gp51/Ab*). After all
incubation procedures the next important task was to select opti-
mal redox mediator for most efcient electron transfer from
carbon rod electrode towards HRP. Several redox active species
were electrochemically tested in 0.05 M phosphate buffer solu-
tion, pH 7.0, containing 5 M of HRP, 0.5 mM of tested redox
mediator and 0.5 mM of hydrogen peroxide. Cyclic voltamme-
try in the potential interval ranging from +500 up to 400 mV
vs. Ag/AgCl was performed by serving bare carbon electrode
Table 2
Comparison of cathodic signals of graphite electrode calculated at 200 mV
from the cyclic voltamperograms (Fig. 1) carried out in the solution of tested
redox mediators, HRP and hydrogen peroxide
Electron donor Current density (A/cm
2
) Relative values of signals (%)
AAP 1.98 17.3
FCA 5.71 49.9
K
4
[Fe(CN)
6
] 4.24 37.1
Pyrogallol 0.18 1.57
TMB 11.43 100
OPD 0.18 1.57
as working electrode in three-electrodes electrochemical circuit.
The same concentrations (0.5 mM) of AAP, FCA, Fe(II), TMB
and OPD were tested in this electrochemical system as potential
redox mediators. Pyrogallol, which is often used in HRP activ-
ity measurements, though is not optically active, but was also
electrochemically tested. For all tested redox mediators the bio-
electrocatalytical currents presented in the corresponding CVs
(Fig. 1) were observed. However, in the cases of OPD, pyrogal-
lol and AAP notably higher current changes due to HRP activity
at positive potential were registered. This potential area was
awkward in the usage of biosensors through probable interfer-
ences. In the CVs performed in the presence of K
4
[Fe(CN)
6
],
TMB and OPD, as a result of HRP catalysis, current changes
at lower potentials were observed. To reduce electrochemical
interference 200 mV was selected as an optimal potential for
constant-potential amperometric detection. Regarding the high-
est current density registered in CVs (Fig. 1) at 200 mV vs.
Ag/AgCl (Table 2) TMB and FCA were found to be the most
efcient redox mediators for further amperometric investiga-
tions. Other authors also have reported TMB (Micheli et al.,
2005; Ionescu et al., 2005; Azedevo et al., 2004) and FCA
(Laschi et al., 2000) as efcient redox mediators for the reg-
istration of HRP-catalyzed reaction. Relatively low potential of
200 mV vs. Ag/AgCl applied for amperometric registration
of HRP activity with redox mediators TMB and FCA was also
an important advantage, since at this potential the background
current in used working solution is relatively small and allows
precise registration of oxidized state of redox mediators.
Changes of cathodic current densityat 200 mVvs. Ag/AgCl
were calculated from the cyclic voltamperograms (Fig. 1)
obtained in the presence of 5 g/ml of HRP, 0.5 mMof hydrogen
peroxide and 0.5 mM of corresponding redox mediator.
3.2. Optimization of amperometric immunoassay
conditions
The aims of optimizations presented in this chapter were:
(i) to test how amperometric signals are depended on HRP
concentration on the surface of the electrode, (ii) to investi-
gate the inuence of nonspecic interaction of HRP on the
surface of electrode. Amperometric immunoassay conditions
were simulated in order to test HRP activity if this enzyme
is conjugated with afnity exhibiting agent (biotin), which is
used to build complexes with avidin-immobilized on the car-
bon rod electrode surface. In this experiment, as described in
B. Kurtinaitiene et al. / Biosensors and Bioelectronics 23 (2008) 15471554 1551
Fig. 1. CVs of the bare graphite electrodes in the 0.05 M phosphate buffer, pH 7.0, containing 0.1 M KCl (black line), with consequently added redox mediators
(0.5 mM, blue line), HRP (5 g/ml, red line) and hydrogen peroxide (0.5 mM, green line). Scan speed 50 mV/s.
the paragraph Simulation of amperometric immunoassay con-
ditions of Section 2: (i) avidin was adsorbed on the bare carbon
electrode surface and then (ii) avidin-modied electrodes were
incubated with various concentrations of the biotin-labeled HRP
(biotinHRP) for 30 min that led to avidin/biotinHRP complex
formation on the carbon electrode surface. Then the steady-
state current generated by HRP-catalyzed enzymatic reaction
was registered in the presence of different concentrations of
H
2
O
2
and redox-mediators (Fig. 2). The steady-state current
obtained corresponds to the maximal velocity of the electrocat-
alytic enzymatic reaction and is proportional to the activity of
the HRP present on the surface of electrode. Consequently, the
steady-state current obtained is proportional to the amount of
biotinHRP, which was bound into avidin/biotinHRP complex
and nonspecically adsorbed on the surface of avidin-modied
carbon rod electrode.
1552 B. Kurtinaitiene et al. / Biosensors and Bioelectronics 23 (2008) 15471554
Fig. 2. Cathodic current vs. concentration of hydrogen peroxide. (Curve 1)
Bare carbon electrode incubated in 11 M biotinHRP solution. (Curves
25) Carbon electrodes modied with avidin were incubated in biotinHRP
solutions containing 0.011, 0.111, 1.11, 11 M of biotinHRP correspond-
ingly. Current was measured at constant 200 mV potential in the presence
of 0.5 mM FCA.
In the control assay, the bare carbon electrodes were treated
with different concentrations of dissolved biotinHRP, and some
unspecic adsorption of the enzyme was observed when the
concentration of biotinHRP exceeded 11 M (Fig. 2, curve
1). For this reason, concentration of biotinHRP for further
tests of avidin-modied carbon electrodes was selected below
11 M. Then avidin-modied carbon electrodes were incubated
in the solutions containing different concentrations of biotin-
labeled HRP and afterwards amperometrically tested in the
presence of 0.5 mM FCA, at electrode potential 200 mV vs.
Ag/AgCl. Consecutive addition of hydrogen peroxide resulted
in signicant increase of cathodic current (Fig. 2). Amperomet-
rically registered steady-state current depends on the surface
concentration of biotinHRP sorbed on the surface of elec-
trode, what is function of biotinHRP concentration in the
solution used for incubation of avidin-modied electrode. The
minimal concentration of biotin-labeled HRP suitable for mod-
ication of carbon electrode was found at 0.011 M (Fig. 2,
curve 2). The maximal steady-state current was registered by
the electrode modied with 11 M biotin-labeled HRP solution
(Fig. 2). The next important kinetic parameter is an apparent
MichaelisMenten constant (K
app
M
), which is correspondingly b
parameter of hyperbolic function y =ax/(b +x) (Ramanavicius et
al., 2005b), where parameter a in this particular case is the max-
imal steady-state current. The K
app
M
of HRP was calculated to
be 0.37 mM for H
2
O
2
. This value is close to the K
app
M
value
(0.4 mM) of HRP reported by Hernandez-Ruiz et al. (2001)
for dissolved HRP. Thus, 0.5 mM concentration of hydrogen
peroxide which exceeded K
app
M
was selected for amperometric
measurements presented in the following sections. In addition
for further experiments blocking stage with albumin solution
was used to avoid nonspecic HRP adsorption on the surface of
electrode.
Fig. 3. BLV specic antibody detection in the BLV-infected sera by standard
ELISA test based on gp51/anti-gp51/HRP-Ab* immune complex formation
and detection of HRP enzymatic activity. The optical density of formed
coloured products vs. dilution of the initial sample (blood serum) containing
anti-gp51 antibodies is presented and approximated by sigmoid dependence:
y =y
0
+a/(1 +(x/x
0
)b), were: y
0
is a background, a is upper value, x
0
is turning
point, and b is escarpment of sigmoid function.
3.3. Detection of gp51, anti-gp51 and HRP-Ab* interaction
by reference methods: AGID test and ELISA
In immunosensor design used immuno-reagents were tested
in order to prove activity of those reagents and to com-
pare sensitivity of newly created immuno-analytical system
with conventional AGID and ELISA assays. In AGID test the
antigenantibody complex forms clear precipitation lines (that
were visible by naked eye) if initial solution of the BLV-infected
serum containing anti-gp51 antibodies was diluted up to 32
times, while any precipitation lines were observed at higher
BLV-infected serum dilutions. Indirect ELISA results using the
same BLV-infected serum were distinctly positive when it was
diluted up to 256 (2
8
) and doubtful at 512 times dilution. The
ELISAresults were estimated by sigmoid function (Fig. 3) were
parameter x
0
, which corresponds x value and at which turn-
ing point of sigmoid curve is detected. In this study, parameter
x
0
was considered as the sensitivity of immuno-analytical sys-
tem, because it represents the x value at which 1/2 (half) of
maximal analytical signal (a y
0
) was detected. For ELISAtest
presented here (Fig. 3) x
0
=391, it means that if initial solution
is diluted by 391 times 1/2 of maximal analytical signal (which
was detected in rst ELISA well) is detected.
3.4. Amperometric immunosensing system
Consecutive incubations in anti-gp51 and HRP-Ab* allowed
formation of the immune complexes gp51/anti-gp51/HRP-Ab*.
Carbon electrodes modied with this immune complex were
tested amperometrically, recording steady-state cathodic current
at 200 mV vs. Ag/AgCl, in PBS (pH 7.0) containing 0.5 mM
H
2
O
2
and 0.5 mM redox mediators TMB or FCA. Experimen-
tal results depicted in Fig. 4 might be well approximated by
sigmoid equation (Ramanaviciene et al., 2006) in cathodic cur-
rents while gp51 modied carbon electrodes were incubated in
B. Kurtinaitiene et al. / Biosensors and Bioelectronics 23 (2008) 15471554 1553
Fig. 4. Calibration plots of immunosensor for anti-gp51 (Ab) obtained in a
sequential immunoassay with FCA (A) and TMB (B) electron donors. The
current was measured amperometrically at constant 200 mV potential in the
presence of 0.5 mM of electron donor and 0.5 mM H
2
O
2
in PBS, pH 7.0 and
presented as current density vs. concentration of analyte.
consecutively diluted anti-gp51 solutions, that is a characteristic
feature for formation of various afnity interaction-based com-
plexes (Rucker et al., 2005). The sensitivity constants (b =0.73,
R=0.9953) for sigmoid curve (Figs. 3 and 4) were found at
608 times antibody dilution for FCA-based system (Fig. 4A)
and b =0.94 at x
0
=2600 times dilution for TMB-based systems
(Fig. 4B). According to the data sensitivity of both electrochemi-
cal systems was slightly better if compared with standard ELISA
kit for detection of anti-gp51 antibodies (where b constant of
sigmoid curve was found 1.19 at x
0
=392 times dilution). The
current density for the same concentrations of analyte for FCA-
based immunosensor was found almost twice higher if compared
with TMB-based immunosensor. However, the sensitivity of
TMB-based amperometric immunosensor was more than four
times higher in comparison with FCA-based immunosensor,
about 80 times higher in comparison with AGID and ve times
higher in comparison with ELISA. Remarkably higher sensitiv-
ity of electrochemical immunosensor was achieved due to the
necessity to dilute initial analyte sample in the ELISA wells. In
ELISAassay presented here initial analyte was 50 times diluted.
Some other authors (Campas and Marty, 2007) reported very
similar sensitivities in competitive ELISA and in corresponding
amperometric immunosensor, which was also based on detec-
tion of HRP activity. It is in agreement with the results of our
present study.
Thereby, both redox mediators might be successfully used in
the design of the amperometric immunosensor for the detection
of antibodies against gp51 and diagnosis of BLV infection.
The investigations of BLV non-infected blood serum sam-
ples performed according to the same protocol gave the current
density in the range of 0.250.3 A/cm
2
and 0.20.25 A/cm
2
if TMB and FIC were applied correspondingly as re-dox medi-
ators. Such current densities correspond to the current densities
registered if 10
4
times diluted BLV-infected blood serum was
investigated. Such high difference in amperometric signals reg-
istered with BLV-positive and BLV-negative samples proves that
here described amperometric method is sensitive enough and
might be applied for detection of anti-gp51 antibodies in real
samples and diagnosis of BLV.
4. Conclusions
The combination of indirect immunoassay technology and
electrochemical methods provided the basis for model system
devoted for the detection of antibodies against gp51. Sim-
ilar systems as single-use immunosensors might be applied
for the detection of other antibodies too. The sensitivity of
here described TMB-based amperometric immunosensor was
more than four times higher in comparison with FCA-based
immunosensor and was about 80 times higher in comparison
with AGID and ve times higher in comparison with ELISA.
Remarkably higher sensitivity of electrochemical immunosen-
sor was achieved due to the necessity to dilute the initial analyte
sample in the rst ELISA well. Such sensitivity is sufcient for
detection of usual anti-gp51 antibody concentration present in
the blood serum of BLV-infected cattle.
Acknowledgement
This work was nancially supported by Agency for Inter-
national Science and Technology Development Programmes in
Lithuania COST action D33.
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