Diagnostics of cattle leucosis by using a biosensor based on the surface plasmon resonance phenomenon
The virus of cattle enzootic leucosis leads to high losses in the rural economy, implies forced slaughter of sick animals, loss of the breed, decrease in productivity, violation of reproduction processes in industrial breeding, and the livestock sector. Among cattle infection diseases, leucosis take...
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| Published in: | Semiconductor Physics Quantum Electronics & Optoelectronics |
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| Date: | 2019 |
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Інститут фізики напівпровідників імені В.Є. Лашкарьова НАН України
2019
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| Cite this: | Diagnostics of cattle leucosis by using a biosensor based on surface plasmon resonance phenomenon / Z.S. Klestova, A.Yu. Yushchenko, Yu.Yu. Dremukh, O. F. Blotska, E.F. Venger, G.V. Dorozinsky, S.O. Kravchenko, Yu.V. Ushenin, N.V. Kachur, V.P. Maslov // Semiconductor Physics Quantum Electronics & Optoelectronics. — 2019. — Т. 22, № 1. — С. 111-118. — Бібліогр.: 27 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860479858744754176 |
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| author | Klestova, Z.S. Yushchenko, A.Yu. Dremukh, Yu.Yu. Blotska, O. F. Venger, E.F. Dorozinsky, G.V. Kravchenko, S.O. Ushenin, Yu.V. Kachur, N.V. Maslov, V.P. |
| author_facet | Klestova, Z.S. Yushchenko, A.Yu. Dremukh, Yu.Yu. Blotska, O. F. Venger, E.F. Dorozinsky, G.V. Kravchenko, S.O. Ushenin, Yu.V. Kachur, N.V. Maslov, V.P. |
| citation_txt | Diagnostics of cattle leucosis by using a biosensor based on surface plasmon resonance phenomenon / Z.S. Klestova, A.Yu. Yushchenko, Yu.Yu. Dremukh, O. F. Blotska, E.F. Venger, G.V. Dorozinsky, S.O. Kravchenko, Yu.V. Ushenin, N.V. Kachur, V.P. Maslov // Semiconductor Physics Quantum Electronics & Optoelectronics. — 2019. — Т. 22, № 1. — С. 111-118. — Бібліогр.: 27 назв. — англ. |
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| container_title | Semiconductor Physics Quantum Electronics & Optoelectronics |
| description | The virus of cattle enzootic leucosis leads to high losses in the rural economy, implies forced slaughter of sick animals, loss of the breed, decrease in productivity, violation of reproduction processes in industrial breeding, and the livestock sector. Among cattle infection diseases, leucosis takes the leading position and comprises 57% of all the other nosological forms, if taking into account the severity of injuries in organs, large-scale character of these diseases, and economic aftermath. This disease can be transferred from animals to men faring with infected milk. The existing methods for cattle vital diagnostics are long-term and weakly sensitive (AGID), or very complex and expensive (ELISA and PCR). In this work, the alternative method for diagnostics, which is the method based on the SPR phenomenon, was proposed. It has been shown for the first time that the SPR method enables to detect antibodies to cattle leucosis virus in the diluted solution (1 vol.%) of weakly positive blood serum taken from sick animals, which cannot be made using the methods AGID and ELISA – in this case the serum is considered as negative, and the tested animal is considered as healthy, although it is carrier of virus.
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ISSN 1560-8034, 1605-6582 (On-line), SPQEO, 2019. V. 22, N 1. P. 111-118.
© 2019, V. Lashkaryov Institute of Semiconductor Physics, National Academy of Sciences of Ukraine
111
Sensors
Diagnostics of cattle leucosis by using a biosensor
based on surface plasmon resonance phenomenon
Z.S. Klestova
1
, A.Yu. Yuschenko
1
, Yu.Yu. Dremukh
1
, O.F. Blotska
1
, E.F. Venger
2
, G.V. Dorozinsky
2
,
S.O. Kravchenko
2
, Yu.V. Ushenin
2
, N.V. Kachur
2
, V.P. Maslov
2
1
State Scientific-Control Institute for Biotechnology and Strains of Microorganisms
30, Donetska str., 03151 Kyiv, Ukraine
E-mail: zinaklestova@gmail.com,
2
V. Lashkaryov Institute of Semiconductor Physics, NAS of Ukraine
41, prospect Nauky, 03680 Kyiv, Ukraine
E-mail: gvdorozinsky@ukr.net, vpmaslov@ukr.net
Abstract. The virus of cattle enzootic leucosis leads to high losses in rural economy,
implies forced slaughter of sick animals, loss of the breed, decrease in productivity,
violation of reproduction processes in industrial breeding and livestock sector. Among
cattle infection diseases, leucosis takes the leading position and comprises 57% of all the
other nosological forms, if taking into account severity of injuries in organs, large-scale
character of these diseases and economic aftermath. This disease can be transferred from
animals to men faring with infected milk. The existing methods for cattle vital diagnostics
are long-term and weakly-sensitive (AGID), or very complex and expensive (ELISA and
PCR). In this work the alternative method for diagnostics is the method based on the SPR
phenomenon was proposed. It has been shown for the first time that the SPR method
enables to detect antibodies to cattle leucosis virus in the diluted solution (1 vol.%) of
weakly positive blood serum taken from sick animals, which cannot be made using the
methods AGID and ELISA – in this case the serum is considered as negative, and the tested
animal is considered as healthy, although it is carrier of virus.
Keywords: bovine leucosis, diagnostic test, surface plasmon resonance.
doi: https://doi.org/10.15407/spqeo22.01.111
PACS 73.20.Mf
Manuscript received 01.02.19; revised version received 14.02.19; accepted for publication
20.02.19; published online 30.03.19.
1. Introduction
Express testing the spread of retroviral infections among
peoples and animals is an important world problem [1].
As known, retroviruses are causative agents for human
secondary immunodeficiency that is observed as AIDS
diseases, they are also the causative agents of the most
dangerous viral diseases that are built-in to the cell
genome, which is inherent, for example, to cattle
enzootic leucosis [2]. It is noteworthy that these diseases
can be transferred from animals to people, for instance,
through use of milk infected with this virus [3].
Numerous publications and official veterinary statistical
data show that, among cattle infection diseases, leucosis
fully dominates, if taking into account severity of organ
injuries, mass manifestation and economical losses, and
comprises 57% as compared with other nosologies [4].
Retroviral infections affect mammals, birds, fishes.
Especially spread is cattle enzootic leucosis that is
registered in all the continents and many countries.
Prophylaxis and elimination of leucosis is very
complicated because of its extremely wide spread. The
special feature of this disease is the long-term incubation
period that lasts without visible abnormalities in health
condition, and this disease has various clinical
presentations. Cattle leucosis virus (CLV) causes
considerable economical losses in agriculture, it forces to
kill sick animals, which leads to losses of breed, lowering
the productivity, breakdown in processes of reproduction
in animal breeding and livestock farming [5, 6].
Up to date, there developed are 20 different
methods for lab diagnostics of leucosis, namely:
haematological, histological and immunological ones that
are based on detection of antibodies induced by viruses
in blood serum of animals. To determine the status of
CLV, the most often used are serological tests [7, 8]. The
main method for intravital diagnostics of CLV is the
reaction of immunodiffusion (AGID) as well as immune-
enzyme analysis (ELISA). Besides, ELISA is used in
problem-free herds of cattle to test the combined trials of
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Klestova Z.S., Yuschenko A.Yu., Dremukh Yu.Yu. et al. Diagnostics of cattle leucosis by using an biosensor based …
112
milk from a group of animals [9]. To investigate
especially valuable animals, there applied is the
polymerase-chain reaction (PCR).
Both methods (AGID and ELISA) are widely
available and relatively cheap ($3-$6 per one test). They
are based on detection of antibodies to viruses. The
deficiency of these methods is their low sensitivity,
therefore, detection of antibodies in animal’s organism
becomes available only for 6–14 weeks after infecting.
Besides, these methods are relatively long (AGID –
72 hours, ELISA – 2 hours) and sometimes give false-
positive results of serological tests for the cattle infected
with CLV and non-infected one. As an alternative, one
can use PCR to detect RNA of CLV, which provides
some advantages over the serological methods.
Application of PCR gives the possibility for early
diagnostics of this infection, however, not all the PCR
methods operate in a similar way, since they have
definite differences in their sensitivity (0.627–0.984) and
specificity (0.89–1.0).
And since this virus can mutate, it is necessary to
choose an individual PCR method for each case of
diagnostics, which is an essential lack. Performing this
test needs, as a rule, 8 hours, and its cost in Ukraine
reaches $30–$50 per test. Detection of virus’ RNA
instead of antibodies as well as potential capability to
detect small concentrations of this virus enables to use
this test for newborn calves, cows in calf and recently
infected animals, when serological tests cannot operate
yet. However, the current cost of these PCR tests is too
expensive for wide application in veterinary practice. The
diagnosis of leucosis is considered to be confirmed under
availability of one of positive results: in serological
investigations with AGID or by using the immune-
enzyme analysis and PCR.
Thus, the known methods of intravital diagnostics
of CLV are either long-lasting and low-sensitive (AGID)
or complicated and expensive (IEA and PCR). The
absence of specific therapy and prophylaxis define
topicality and priority of the problem of cattle leucosis
not only for veterinary medicine but for biology as a
whole. Therefore, development of diagnostic methods
enabling to detect diseases at their initial stages and in
the shortest terms is an extremely important and urgent
task.
One of these diagnostic methods is the method
based on the phenomenon of surface plasmon resonance
(SPR). As compared with the traditional methods, this
one has the following weighty advantages: capability to
study processes of molecular interaction in nano-sized
layers in a real time scale, small volume of the sample of
studied substance (less than 10 µL), absence of the
necessity to use special markers or fluorescent lables for
the studied substance (analyte) [10]. The devices based
on SPR have biological sensors consisting of a sensitive
element and physical transducer.
The sensitive element is a thin metal layer
(40…60 nm) on the surface of dielectric substrate. In the
majority of SPR devices, this metal layer consists of gold
due to its high chemical inertness. The biological sensor
on the surface of sensitive element contains biologically
active substance (receptor) that selectively reacts only
with respective analyte via covalent bonding that is also
named as specific interaction. But really in any biological
reactions, non-specific interaction takes place, when
there present are partial filling the surface of sensitive
element and domination of physical adsorption over
covalent bonding, which lowers both sensitivity and
selectivity of SPR devices.
To minimize non-specific adsorption on the surface
of sensitive element, usually applied is adsorption of
functional groups to immobilize receptors
complementary to them. The most successful methods to
functionalize the gold surface are based on the
conception of molecular self-assembling thyols or
disulphide molecules [11]. Development of highly stable
receptors [12] enabled to perform effective testing the
medicinal preparations by using the SPR method both in
the process of their production and usage. SPR devices
are widely used in detection of bacteria and viruses,
namely: Staphylococcus aureus [13, 14], Pichia pastoris
[15], Leishmania [16], Mycobacterium tuberculosis [17],
human adenovirus [18], Epstein-Barr virus [19], bird’s
flu virus H5N1 [20], dengue virus [21], hepatitis B and
AIDS [22, 23].
In [24], the authors have demonstrated non-invasive
diagnostics of CLV by using the immunosensor analysis
with the SPR method. Application of this method to cow
lactoserum enabled to detect antibodies to proteins p24,
pg51 of CLV, presence of which indicates development
of the disease in animals. Interaction of antigens (the
proteins p24, pg51 of CLV) with specific to them
antibodies is registered as changes in the resonance angle
of SPR. The authors of this approach use the
modification of SPR sensor construction with the
sensitive element from gold layer without any functional
coating, i.e., without special preparation of the metal
surface in this sensitive element.
Non-uniformity of the metallic layer in the sensitive
element causes only partial interaction over the total
surface of its area as well as presence of non-specific
adsorption on the surface of the element, which defines a
negative effect both on sensitivity and reliability of
results obtained using the SPR method. This non-
uniformity was caused, first of all, by the method of gold
layer deposition, namely, vacuum sputtering. Therefore,
we performed additional investigations that showed the
necessity of specific preparation of the metal layer
surface in the sensitive element to improve adhesion
(immobilization) of the receptor.
Being based on these investigations, we developed a
new way to produce the sensitive elements of SPR
sensors. This way implies the annealing of sensitive
elements with the preliminary deposited gold layer of the
thickness 50±2 nm at the temperatures 100…140 °С for
10 to 40 min and following irradiation with UV light of
the wavelength 205…315 nm also for 10…40 min [25].
It provided to reduce non-uniformity of the sensitive
element surface as well as improve, in what follows, its
functionalization and to reduce non-specific adsorption
SPQEO, 2019. V. 22, N 1. P. 111-118.
Klestova Z.S., Yuschenko A.Yu., Dremukh Yu.Yu. et al. Diagnostics of cattle leucosis by using an biosensor based …
113
on its surface. Applied in practice are sensitive elements
of SPR sensors, which are preliminarily functionalized
surface [26]. Some deficiency of this technology is an
additional loss of time for activation of the functionalized
surface and immobilization of the receptor on it. In
essence, these operations are only preparation
procedures, since the result of diagnostics is determined
analyzing observed interaction of the receptor with
analyte. In our opinion, an optimal way to reduce the
time spent on diagnostics is to combine these preparation
procedures of functionalization, activation and
immobilization of the receptor.
In this work, we performed investigations of the
influence of functionalization applied to the gold surface
of sensitive elements in SPR sensors as well as
preliminary antigen immobilization on the sensitivity of
sensors when diagnosing cattle enzootic leucosis.
2. Experimental investigations
Studying the efficiency of gold surface functionalization
and its influence on the sensitivity of SPR sensors was
made with the SPR device “Plasmon-6” (Fig. 1) deve-
loped in the V. Lashkaryov Institute of Semiconductor
Physics, NAS of Ukraine [11].
With this aim, we deposited the receptor – cattle
antigen on the metal surface of sensitive elements. Then,
we analyzed interaction of the receptor with analyte
(i.e., antibody to CLV) with account of the relative shift
observed for the minimum of the SPR resonant
characteristics.
To interpret the results of investigations, in
addition, we carried out testing the blood serum specific
to CLV with antibodies by using the AGID method in the
State Scientific-Control Institute for Biotechnology and
Strains of Microorganisms (SSCIBSM).
To perform the above investigations, we used the
following materials and reagents: re-inoculated cell
culture of follicular lymphoma prepared from the sheep
embryonic kidney FLK-BLV (FLK-SBBL, FLK 50/100,
FLK 71, depositary at the National Center for
Microorganism Strains at SSCIBSM), cattle antigen
(cryobank of cell cultures at SSCIBSM), cow blood
Fig. 1. External look of the device “Plasmon-6” with the SPR
sensor.
serum (husbandries of the Poltava region, Ukraine),
nutrient medium “DMEM High Glucose”, w/L –
Glutamine, w/o – Sodium Pyruvate, Sterile Filtered
(Biowest, series S17885L0102, USA), cattle fetal blood
serum “Fetal Bovine Serum Premium” (Biowest, series
S14317S181B, USA), trypsin “Trypsin 0,25%”, w/o
Calcium, w/o Magnesium, w/o Phenol Red, Sterile
Filtered (Biowest, series S16067L0910, USA), 0.02%
versene solution (SSCIBSM), antibiotic “Gentamycin”
(4% solution for injections, pharmaceutic company
“Zdorovya” Ltd, Ukraine) Triton X-100 (Merck, USA),
11-mercaptoundecan acid (Merck, USA), 11-
mercaptoundecanol (Merck, USA), absolute methanol
(“Khimlaborreaktive” Ltd, Ukraine), N-ethyl-N′-
dimethyl-amine-propyl-carbo-diimide hydrochloride
(EDC) (Merck, USA), N-hydrosuccinimide (NHS)
(Merck, USA), physiological solution (“Pharmak” Ltd,
Ukraine), twice distilled water (SSCIBSM).
Being aimed at unification and harmonization of
methods for controlling the diagnostic ways in accord
with international regulations and norms, developed in
SSCIBSM was the National Standard for Leucosis Sera
intended for standardization of ways targeted on
serological diagnostics of enzootic CVL – “The set of
blood sera for standardization of cattle leucosis virus
antigen by using the reaction of immunediffusion
(AGID)”, registration certificate №ВВ-00664-06-13.
With this aim, we received a set of cattle blood sera from
the international reference lab studying enzootic bovine
leucosis. This set is usually used as the reference one in
the world practice to estimate quality of leucosis test-
systems.
The re-inoculated culture of cells FLK was
cultivated for 7 days in a special culture medium
consisting of nutrient solution DMEM with addition of
10% cattle fetal blood serum preheated for 30 min at a
temperature of 56 °С. Replication of the leucosis virus
was not accompanied by destruction of the cell
monolayer, which corresponds to the main characteristics
and norms of this culture. Then, supermatant liquid of the
FLK cell culture that contains this virus was centrifuged
and processed with de-ionized surface-active substance
Triton X-100 to avoid adsorption of antigen on the walls
of vessels. The concentrated and inactivated virus antigen
was used to study its activity in AGID. With this aim, we
prepared twofold dilutions of antigen (1:2…1:16), which
were used with the standard blood sera. For
measurements by using the SPR method and the device
“Plasmon”, we prepared dilutions of sera in physiological
solution of рН 7.4 that contained 0.14 М of sodium
chloride.
The sensitive elements of the device “Plasmon-6”
were made in accord with the way described in [25]. To
perform the experiments, we produced two groups of
sensitive elements of five unities per each group with
functionalized and non-functionalized metal surface. To
provide functionalization, these sensitive elements were
dipped one time into the mixture of following solutions:
11-mercapto-undecan acid (0.88±0.01 mg/cm3) in
absolute methanol (20 vol.%) and 11-mercapto-
SPQEO, 2019. V. 22, N 1. P. 111-118.
Klestova Z.S., Yuschenko A.Yu., Dremukh Yu.Yu. et al. Diagnostics of cattle leucosis by using an biosensor based …
114
undecanol (0.22±0.01 mg/cm3) in absolute methanol
(60 vol.%) and were kept in an airproof glass vessel at
the temperature 40±1 °С for 6 hours.
After this keeping, the sensitive elements were first
washed out in absolute methanol and then with distilled
water by dipping them in Petri dish filled with these
solutions by keeping there for 15 min in each solution. In
what follows, the surface of sensitive elements was
activated with the mixture of water solutions EDC and
NHS by dipping them into this mixture in Petri dish and
keeping there for 15 min. Then, using a micropipette we
coated the surface with 50 µL of CLV antigen solution,
kept at the temperature 20 °С and later dried at the
temperature 37 °С for 20 min. After that, the sensitive
elements with immobilized and dried antigen were
placed into the measuring double-channel cuvette inside
“Plasmon-6” with the volume 5 µL per channel. This
cuvette was equipped with inlet and outlet fittings for
each channel to remove studied liquids (distilled water
and CLV antibodies). By turn, these liquids were pumped
through one channel of the measuring cuvette with the
peristaltic pump providing the velocity 10 µL/min for
CLV antibody solutions and 50 µL/min – for buffer.
This velocity was chosen to minimize the pressure
difference in the cuvette and provide a laminar liquid
flow. This stage was followed by pumping the solution of
antibodies through the cuvette to provide their binding
with the receptor. The second channel was used as the
reference one to compensate the influence of external
factors, such as changes in the temperature of ambient
medium. It was pumped through with the physiological
solution. All the measurements were performed at the
room temperature (22±1 °С).
To choose optimal duration suitable for
immobilization of antigen-receptor on the functionalized
surface of the sensitive element, we studied two cases:
long-term immobilization (22 hours) and the mean-term
one (60 min). The period of interaction between antigen
and antibody was determined experimentally and equaled
36 min: this time was sufficient for binding of antigens
with antibodies over the whole surface of the sensitive
element in the SPR sensor, which was confirmed by the
absence of any shift of the resonant characteristics after
30-min interaction (i.e., completion of the process). The
processes of activation of the sensitive element surface
(with EDC/NHS), of cattle antigen immobilization and
interaction antigen-antibody were observed as respective
angular shifts of the minimum inherent to the SPR
resonance characteristics in time when pumping the
necessary reagents through the measuring cuvette.
3. Results
To carry out the investigations by using the SPR method,
we selected the samples of antigen that demonstrated a
clear line of precipitation in AGID (Fig. 2). These
samples were additionally cleaned using ultra-filtration
and adsorption on porous glass with the pore diameter
100 nm. In the experiments, we used 10-fold dilution of
antigen in distilled water. All the sera were preliminary
Fig. 2. Immunediffusion reaction of cattle antigen (central
lunula) with the National standard samples of blood sera
(positive – 100%, weakly positive – 10%, negative) and
positive reference – peripherical lunulae.
tested in SSCIBSM and conditionally separated in three
groups, namely: AGID-positive, AGID-negative and the
doubtful ones, i.e., those non-providing in AGID a clear
precipitation line in agar gel.
Long-term immobilization of antigen on
functionalized and non-functionalized surfaces of
sensitive element was carried out for 22 hours. When
doing so, the antigen solution was cyclically pumped
through the measuring cuvette. If studying interaction
“antigen–antibody”, we additionally used the weakly
positive 10-fold diluted serum (10 vol.%), since the 100-
fold diluted serum could provide a signal close to the
level of noise.
Fig. 3 shows the plot of kinetics corresponding to
interaction “antigen – antibody” for two solutions of
weakly positive sera (1 vol.% and 10 vol.%) in the
process of long-term antigen immobilization on the
surface of sensitive element with the functional coating
and without it. The ordinate axis represents values of
angular shifts of the SPR resonance characteristic
minimum in angular degrees. As seen from the figure,
when substituting biomaterials in the cuvette channels,
one can observe the respective angular shift of the
resonance minimum as a result of changing the refraction
index at the surface of SPR sensor sensitive element.
The kinetics of surface activation and antigen
immobilization are not shown in this figure, since these
processes were not measured through their considerable
total duration. The measurements started from washing
out the cuvette channels with physiological solution (the
tenth minute of kinetics). At the beginning of 25th
minute, this physiological solution was substituted with
1-% serum solution that was pumped through for
100 min to reach saturation (110th min). Then, the
measuring channels were again washed out with the
physiological solution. Starting from the 160th min of
kinetics, the cuvette was filled with the higher
concentration (10 vol.%). Like to the previous case, the
serum was pumped through for 100 min, and then the
cuvette was washed out with the physiological solution.
SPQEO, 2019. V. 22, N 1. P. 111-118.
Klestova Z.S., Yuschenko A.Yu., Dremukh Yu.Yu. et al. Diagnostics of cattle leucosis by using an biosensor based …
115
Fig. 3. Kinetics of interaction “antigen–antibody” for two
solutions of weakly positive sera (1 and 10 vol.%) after long-
term (22 hours) immobilization of antigen on the surface of
sensitive element with the functional coating and without it.
Fig. 4. Kinetics of interaction “antigen–antibody” on the
surface of functionalized sensitive element for mean-time
antigen immobilization (60 min) of the weakly positive
serum with antibody in solution 100-fold diluted (Serum 1%)
in the physiological solution. The SPR sensor response equals
∆θ = 144±3 arcsecond.
In both cases of concentration, the physiological
solution could not wash out the antibody in full volume,
which indicated the presence of interaction, i.e., covalent
binding between antigen and antibody. In the case of
sensitive element without functional coating, the sensor
response was considerably lower. It is related with the
fact that the majority of antigen-receptors on the surface
occupied wrong space position, and their sites for
interaction with antibody were used for interactions
“antigen–antigen” as well as “antigen–alkanthiol”.
The angular shift of resonant characteristic for the
sensitive element with the functional coating (Fig. 4) in
the case of weakly positive serum with antibody, which
was 100-fold diluted (1 vol.%), was 9-fold higher
(∆θ = 155±3 arcsecond) than for the sensitive element
without any functional coating (∆θ = 17±3 arcsecond). In
the case of higher concentration of the 10-fold diluted
weakly positive serum (10 vol.%), the angular shift of
resonance characteristic for the sensitive element with the
functional coating was 8-fold higher
(∆θ = 1454±3 arcsecond) than that for the sensitive
element without functional coating, which was
∆θ = 187±3 arcsecond.
Shown in Fig. 4 is the kinetic of the SPR sensor
response to the reaction of interaction “antigen–
antibody” on the surface of functionalized sensitive
element for mean-time duration of antigen
immobilization. The ordinate axis represents a relative
shift of resonant characteristic minimum, which is the
difference between signals in the measuring and
reference channels. It provides compensation of the
temperature drift of the resonant characteristic minimum
that could occur, since the temperature factor has an
essential influence on results of measurements [27]. For
instance, the change in distilled water temperature by
0.25 °С results in the change of its refractive index by the
value 2·10–5, which is commensurate by its value with
the detection limit of the device “Plasmon-6” (2·10–5).
The procedures of washing out, activation, repeated
washing and antigen immobilization with the following
washing out in the physiological solution are not shown
in this plot, since the increase in sensitivity is only
determined with account of responses to interaction
“antigen–antibody”. It is seen from this plot that after the
187th min substitution of the physiological solution by
that of serum with antibody begins, and after the 190th
min there begins the proper reaction of covalent binding
between antigen and antibody.
Starting from the 224th min, we made washing out
the cuvette with the physiological solution. As a result,
the antibodies that were not bound with antigens were
washed out, which was confirmed by the decrease in the
relative shift of the resonant characteristic minimum to
the value higher than the initial one inherent to the 180th
min of measurements.
When immobilization lasted for 60 min, in the case
of weakly positive serum with antibody in 100-fold
dilution (1 vol.%), the angular shift of resonant
characteristic inherent to the sensitive element with
functional coating was ∆θ = 144±3 arcsecond, and for the
element without this coating it was ∆θ = 18±3 arcsecond.
Thus, when antigen immobilization lasts one hour,
availability of the functional coating enhanced the
sensitivity of the SPR sensor by 8 times, as compared
with the sensitive element without this coating.
4. Discussion
Despite the long-term immobilization of the sensitive
element surface without functional coating (Fig. 3),
which provided filling all its surface, the response was
relatively low (∆θ = 17±3 arcsecond). It can be explained
as follows: the majority of receptors (near 80%) took up
such a position in space that their interaction with
antibodies became impossible.
Time of measurement, min
Time of measurement, min
SPQEO, 2019. V. 22, N 1. P. 111-118.
Klestova Z.S., Yuschenko A.Yu., Dremukh Yu.Yu. et al. Diagnostics of cattle leucosis by using an biosensor based …
116
5. Conclusions
The virus of cattle enzootic leucosis leads to high losses
in rural economy, implies forced slaughter of sick
animals, loss of the breed, decrease in productivity,
violation of reproduction processes in industrial breeding
and livestock sector. Among cattle infection diseases,
leucosis takes the leading position and comprises 57% of
all the other nosological forms, if taking into account
severity of injuries in organs, large-scale character of
these diseases and economic aftermath. This disease can
be transferred from animals to men faring with infected
milk. The existing methods for cattle vital diagnostics are
long-term and weakly-sensitive (AGID), or very complex
and expensive (ELISA and PCR).
The alternative method for diagnostics is the
method based on the SPR phenomenon, but it is efficient
and reliable only in the case of functionalization of the
sensitive element surface in the SPR sensors. It has been
demonstrated experimentally that application of this
coating enhances the sensitivity of these sensors at least
8-fold, as compared with the sensors without this coating.
In this case, duration of diagnostics is only 4 hours and
can be made shorter (for 2 hours) by respective
optimization of this procedure. It has been shown for the
first time that the SPR method enables to detect
antibodies to cattle leucosis virus in the diluted solution
(1 vol.%) of weakly positive blood serum taken from sick
animals, which cannot be made using the methods AGID
and ELISA – in this case the serum is considered as
negative, and the tested animal is considered as healthy,
although it is carrier of virus.
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Authors and CV
Yuschenko Alla – young scientist,
biologist of the 1st category. She
works at the Department of
Biotechnology and Quality
Controlling of Viral Preparations of
SSCIBSM. She graduated from
Taras Shevchenko Kyiv National
University in 2014. In SSCIBSM,
she works since 2016.
Prof., Dr. Zinaida Klestova
In 1981, graduated from Taras
Schevchenko National Kiev Univer-
sity, Biology Faculty, specialty in
genetic and virology. She possesses
the academic degree of the Doctor
of Veterinary Sciences and PhD of
Biological Sciences and the
academic status of the senior
researcher (1993). At the State Scien-
tific-Control Institute of Biotechnology and Strains of
Microorganisms, she is a Deputy Director of Scientific
Work and the Head of scientific coordination of
biosafety and biorisks Department. She possesses
considerable experience of the research work and leading
scientist in the spheres of biosecurity, biorisks, virology,
microbiology, bacteriology, immunology and genetics.
E-mail: zinaklestova@gmail.com
ORCID: https://orcid.org/0000-0003-0771-7808
Dremuh Julia,
Ph.D. student. Education –
graduated from Veterinary college
in 2008 by the specialty Veterinary
Medicine. In 2008, she entered the
higher education institution –
National University of Life and
Environmental Sciences of Ukraine
and attained complete higher educa-
tion by the specialty Veterinary Medicine. In 2012,
Dremuch entered a PhD program at the Institute of
Veterinary Medicine of NAAS by the specialty 16.00.03
– Veterinary Microbiology, epizootiology, infectious
diseases and immunology. She works at the laboratory
of virology and antiviral drugs.
E-mail: dremych@gmail.com
Oksana Fedorivna Blotska –
DVM, PhD, Head of the Division of
Check and Industrial Virus Strains
Support of State Scientific Control
Institute of Biotechnology and
Strains of Microorganisms, Kyiv,
Ukraine. She possesses considerable
experience of the research work and
is a skilled scientist in the spheres of
virology, microbiology, immunolo-
gy, serology, biosafety, biosecurity and biorisks. She was
the supervisor of 2 State budget 3-years scientific
programs, manager of 2 and member of one
International Projects. She has published 139 scientific
articles, 3 patents pending, created 7 new
immunobiological means and 8 new National standards.
E-mail:blotskaya@ua.fm
SPQEO, 2019. V. 22, N 1. P. 111-118.
Klestova Z.S., Yuschenko A.Yu., Dremukh Yu.Yu. et al. Diagnostics of cattle leucosis by using an biosensor based …
118
Dr. Yevgen Venger, Head of the
scientific department, Correspon-
ding Member of NAS of Ukraine,
Dr of Sciences, Professor. He has an
experience of managing the State
scientific and technical program for
development of sensor devices in
2008 – 2017, projects by the State
order and innovation ones.
E-mail: vengeref@gmail.com
Glib Dorozinsky got his degree MS
in Physics and Techniques (Elect-
ronic home appliances) at the Natio-
nal Technical University of Ukraine
“Kiev Polytechnic Institute”, Radio-
technical faculty (Ukraine) in 2012.
Since 2016 PhD, senior researcher at
the V. Lashkaryov Institute of Semi-
conductor Physics, NAS of Ukraine.
His research interests lie in physics of surfaces, develop-
ment and design chemical sensors and biosensors for
applying in different fields like medicine, pharmacology,
industry and ecology.
E-mail: gvdorozinsky@ukr.net
ORCID: https://orcid.org/0000-0002-7881-2493
Serhii Kravchenko, Senior
researcher, PhD.
Graduated from Lviv National
Technical University, specializing in
technology engineer, chemical
technology of biologically active
compounds. Graduated from the
Institute of Bioorganic Chemistry of
the National Academy of Sciences
of Ukraine. Since 2006, he works at
the V. Lashkaryov Institute of Semiconductor Physics
NAS of Ukraine, currently he holds the position of
Senior Researcher. The field of scientific interests is the
development of sensitive elements of physical
converters (QCM, SPR, SERS), obtaining plasmon and
fluorescent nanoparticles for sensory applications.
E-mail: kravchenko.srg@gmail.com
Yurii Ushenin got his degree in
Vision and spectroscopy at Taras
Shevchenko National University of
Kyiv. He graduated from the Kiev
University, Physics department
(Optical instruments and spectro-
scopy) in 1971. Since 1973 he was
with the Institute of Semiconductor
Physics of National Academy of Sciences of Ukraine as
an engineer and a senior researcher (since 1992 up to
now). His areas of expertise include systems of
collection and data processing, software design (Pascal,
Delphi) and optical devices including SPR and planar
semiconducting interferometer. E-mail: ushyury68@i.ua
Nataliya Kachur got her degree MS
in Physics and Techniques at the
National Aviation University of
Ukraine, Mechanical faculty
(Ukraine) in 2006.
Her research interests in physics of
surfaces, development and design of
sensors for application in control of
quality of transparent materials.
E-mail: natalykachur@gmail.com
ORCID: https://orcid.org/0000-0001-6868-8452
Dr. Volodymyr Maslov got his
degree in Physics and Technics at
the Moscow Institute of Physics and
Technology State University
(Russia) in 1969, his PhD in
Material Science at the Moscow
Institute of Physics and Technology
State University (Russia) in 1973.
Doctor of Material Science since
2009, Honored inventor of Ukraine.
He is professor of National Technical University of
Ukraine “Kyiv Polytechnic Institute” since 2010. His
research interests in several topics of optical engineering
and physical behavior of functional materials and
phenomena of surface plasmon resonance and
application of it in medicine and ecology now.
E-mail: vpmaslov@ukr.net
ORCID: https://orcid.org/0000-0001-7795-6156
|
| id | nasplib_isofts_kiev_ua-123456789-215419 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1560-8034 |
| language | English |
| last_indexed | 2026-03-23T18:50:57Z |
| publishDate | 2019 |
| publisher | Інститут фізики напівпровідників імені В.Є. Лашкарьова НАН України |
| record_format | dspace |
| spelling | Klestova, Z.S. Yushchenko, A.Yu. Dremukh, Yu.Yu. Blotska, O. F. Venger, E.F. Dorozinsky, G.V. Kravchenko, S.O. Ushenin, Yu.V. Kachur, N.V. Maslov, V.P. 2026-03-16T10:58:17Z 2019 Diagnostics of cattle leucosis by using a biosensor based on surface plasmon resonance phenomenon / Z.S. Klestova, A.Yu. Yushchenko, Yu.Yu. Dremukh, O. F. Blotska, E.F. Venger, G.V. Dorozinsky, S.O. Kravchenko, Yu.V. Ushenin, N.V. Kachur, V.P. Maslov // Semiconductor Physics Quantum Electronics & Optoelectronics. — 2019. — Т. 22, № 1. — С. 111-118. — Бібліогр.: 27 назв. — англ. 1560-8034 PACS: 73.20.Mf https://nasplib.isofts.kiev.ua/handle/123456789/215419 https://doi.org/10.15407/spqeo22.01.111 The virus of cattle enzootic leucosis leads to high losses in the rural economy, implies forced slaughter of sick animals, loss of the breed, decrease in productivity, violation of reproduction processes in industrial breeding, and the livestock sector. Among cattle infection diseases, leucosis takes the leading position and comprises 57% of all the other nosological forms, if taking into account the severity of injuries in organs, large-scale character of these diseases, and economic aftermath. This disease can be transferred from animals to men faring with infected milk. The existing methods for cattle vital diagnostics are long-term and weakly sensitive (AGID), or very complex and expensive (ELISA and PCR). In this work, the alternative method for diagnostics, which is the method based on the SPR phenomenon, was proposed. It has been shown for the first time that the SPR method enables to detect antibodies to cattle leucosis virus in the diluted solution (1 vol.%) of weakly positive blood serum taken from sick animals, which cannot be made using the methods AGID and ELISA – in this case the serum is considered as negative, and the tested animal is considered as healthy, although it is carrier of virus. en Інститут фізики напівпровідників імені В.Є. Лашкарьова НАН України Semiconductor Physics Quantum Electronics & Optoelectronics Sensors Diagnostics of cattle leucosis by using a biosensor based on the surface plasmon resonance phenomenon Article published earlier |
| spellingShingle | Diagnostics of cattle leucosis by using a biosensor based on the surface plasmon resonance phenomenon Klestova, Z.S. Yushchenko, A.Yu. Dremukh, Yu.Yu. Blotska, O. F. Venger, E.F. Dorozinsky, G.V. Kravchenko, S.O. Ushenin, Yu.V. Kachur, N.V. Maslov, V.P. Sensors |
| title | Diagnostics of cattle leucosis by using a biosensor based on the surface plasmon resonance phenomenon |
| title_full | Diagnostics of cattle leucosis by using a biosensor based on the surface plasmon resonance phenomenon |
| title_fullStr | Diagnostics of cattle leucosis by using a biosensor based on the surface plasmon resonance phenomenon |
| title_full_unstemmed | Diagnostics of cattle leucosis by using a biosensor based on the surface plasmon resonance phenomenon |
| title_short | Diagnostics of cattle leucosis by using a biosensor based on the surface plasmon resonance phenomenon |
| title_sort | diagnostics of cattle leucosis by using a biosensor based on the surface plasmon resonance phenomenon |
| topic | Sensors |
| topic_facet | Sensors |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/215419 |
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