Accepted Manuscript

Title: Application of oriented and random antibodyimmobilization methods in immunosensor design

Author: <ce:author id="aut0005" biographyid="vt0005">Julija Baniukevic<ce:author id="aut0010"biographyid="vt0010"> Justina Kirlyte<ce:authorid="aut0015" biographyid="vt0015"> ArunasRamanavicius<ce:author id="aut0020"biographyid="vt0020"> Almira Ramanaviciene

PII: S0925-4005(13)00418-8DOI: http://dx.doi.org/doi:10.1016/j.snb.2013.03.126Reference: SNB 15351

To appear in: Sensors and Actuators B

Received date: 30-9-2012Revised date: 27-3-2013Accepted date: 28-3-2013

Please cite this article as: J. Baniukevic, J. Kirlyte, A. Ramanavicius, A.Ramanaviciene, Application of oriented and random antibody immobilizationmethods in immunosensor design, Sensors and Actuators B: Chemical (2013),http://dx.doi.org/10.1016/j.snb.2013.03.126

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Application of oriented and random antibody immobilization methods

in immunosensor design

Julija Baniukevic, Justina Kirlyte, Arunas Ramanavicius, Almira Ramanaviciene*

NanoTechnas - Center of Nanotechnology and Materials Science, Faculty of Chemistry, Vilnius

University, Naugarduko 24, LT-03225 Vilnius, Lithuania

Corresponding author:

Almira Ramanaviciene. E-mail: almyra@imi.lt, phone: +370 67203653, Fax: +370 52330987.

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Application of oriented and random antibody immobilization methods

in immunosensor design

Abstract

An immunosensor for the determination of bovine leukemia virus (BLV) antigen (gp51) using surface

plasmon resonance (SPR) equipment and chip (SPR-chip) modified by oriented and randomly

immobilized antibodies (anti-gp51) was developed. Different conditions of anti-gp51 reduction by 2-

mercaptoethanol (2-ME) and dithiothreitol (DTT) solutions were compared and explored. The best

gp51 detection sensitivity was monitored using SPR-chip modified with oriented antibody fragments

(frag-anti-gp51) obtained after reduction with DTT at 50 mM concentration. The SPR sensor could

detect gp51 antigen in a range from 0.01 to 0.5 mg/mL. The developed SPR sensor offered the limit of

detection (LOD) as low as 0.0028 mg/mL, while the limit of quantification was as low as

0.0092 mg/mL with very good repeatability during the three detection–regeneration cycles (1-4 %).

Oriented immobilization of frag-anti-gp51 was found to be more suitable for the design of SPR-

immunosensor for the detection of gp51 if compared with random immobilization of anti-gp51. This

method was successfully applied for the detection of gp51 in blood serum samples in a rapid, reliable

and selective manner.

Keywords: SPR, immunosensor, antibody immobilization, BLV.

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1. Introduction

Recently the SPR method has become one of the main methods for the investigation and

determination of materials based on affinity interaction. SPR biological sensors increasingly are used

for the determination of the new drugs characteristics [1], diagnosis of virus induced diseases [2],

detection of toxins [3], allergens [4], hormones, steroids, and immunoglobulins [5], for the

evaluation of food quality and safety [2], environment control [6], characterization of antibodies and

quantitative immunoanalysis [7,8]. SPR biological sensors are capable to detect unlabeled analytes

in real-time by registering the formation of ligand-receptor complex [9], which allows detailed

investigation of biological molecules interaction kinetics [10]. Therefore, SPR method is currently

one of the most promising ligand-receptor, antigen-antibody detection methods [11,12], which

increasingly is used in disease diagnosis and biomedical research.

Retroviruses are known as agents which cause various malignant and non-malignant diseases

in animal over a wide range of species. Bovine leukemia virus (BLV) is an oncogenic retrovirus and

the major target cells of BLV are B-lymphocytes. BLV cause a nonneoplastic, persistent

lymphocytosis, and a neoplastic proliferation of B lymphocytes, enzootic bovine leukosis. Recently,

the European Economic Community declared most of its member states as free from enzootic bovine

leucosis. However, BLV is still wide disseminated in USA [13], Argentina [14], Japan [15] and other

regions of the world. Due to the BLV is one of the most common infectious viruses of cattle and is

endemic in many dairy herds, control and eradication programs based on early detection of infected

cattle and subsequent culling face a major economic task. It is expected that the traditional methods

give way to higher performance, more sensitive and faster methods such as SPR immunosensors.

Immunosensors are based on the specific recognition between antibodies and antigens when

one of interacting components is immobilized on the surface. Antibodies can be immobilized onto

different solid surfaces by physical or chemical adsorption, covalent coupling, using defined

linkages such as glutaraldehyde, carboiimide, or gold nanoparticles, proteins A and G [16,17,18].

Oriented immobilization of the antibody on the surface of a sensor chip is one of the most important

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criteria for the improvement of the sensitivity and specificity of immunosensors [19,20]. Number of

methods for oriented immobilization of biomolecules, including immobilization through the

biotin/(strept)avidin complexes, his-tagged proteins onto Ni2+-chelating surfaces [21], proteins A and

G were reported [22]. Several methods have been reported for the oriented immobilization of

antibody fragments. Oriented immobilization of antibody fragments leads to much higher antigen

binding capacity, sensitivity of immunosensor and better ability to regenerate the surface, but does

not cause substantial decrease or significant distribution of the apparent affinity constants [9,23,24].

Furthermore, high operational stability of gold-electrodes modified by fragmented-antibodies makes

them very attractive and applicable for immunoanalytical purposes [1,19].

Thus, to develop high performance, sensitive and reproducible immunosensor for the detection

of target molecules, the appropriate immobilization of antibodies must be carried out. In this study

oriented and random antibody immobilization methods in SPR immunosensor design were

compared. The obtained fragments of antibodies can be self-assembled on gold surface due to the

thiol group (-SH) without any additional reagents or self-assembled monolayers. The amount of the

immobilized antibodies or their fragments and antigen detection efficiency were investigated and

compared using SPR.

2. Experimental part

2.1. Materials

Bovine leukemia virus (BLV) protein gp51 and bovine blood serum containing antibodies (anti-

gp51) specifically interacting with antigen gp51 were obtained from BIOK (Kursk, Russia). Bovine

serum albumin (BSA) 1,4-dithiothreitol (DTT), and sodium dodecylsulphate (SDS) were purchased

from Carl Roth (Germany). Unstained protein molecular weight marker was purchased from Thermo

Fisher Scientific - Fermentas (Vilnius, Lithuania). Refractive index matching fluid (n=1.518) was

obtained from Cargille Labs (Cedar Grove, New Jersey). 0.01 M phosphate buffered saline (PBS),

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pH 7.4, was received from Medicago (Sweden). N-(3-dimethylaminopropyl)-N-ethylcarbodiimide

hypochloride (EDC), N-hydroxysuccinimide (NHS), 11-mercaptoundecanoic acid (11-MUA),

ethanolamine, 2-mercaptoethanol (2-ME) and other chemicals of analytical-reagent grade or even

better were obtained from Sigma-Aldrich (Steinheim, Germany). All aqueous solutions were

prepared using HPLC-grade water purified in a Purator-B Glas Keramic (Berlin, Germany).

2.2. Anti-gp51 precipitation using ammonium sulphate

Serum containing anti-gp51 was mixed and continuously stirred with an equal amount of saturated

solution of ammonium sulphate, which was added dropwise to prevent local precipitation of

proteins. The resulting mixture was kept at 4ºC temperature overnight. Then the solution was

centrifuged at 5000 rpm for 20 min. The supernatant was decanted and the pellet was dissolved in

PBS, pH 7.4. The purification procedure was performed twice. The solution containing specific anti-

gp51 antibodies was dialyzed against the PBS at 4ºC overnight. Finally, the immunoglobulin

concentration was estimated using UV-Vis spectrophotometer (Lambda 25, PerkinElmer, USA) at

λ=260 nm and λ=280 nm. Determined immunoglobulin concentration after purification was

5.69 mg/mL.

2.3. Preparation of frag-anti-gp51

Frag-anti-gp51 were obtained by chemical reduction of intact antibodies using DTT or 2-ME agents.

Particular volumes of PBS, anti-gp51 (2 mg/mL) solution and 10 mM 2-ME (final concentration in

the reaction solution 15 mM, 35 mM or 70 mM) or 10 mM DTT (final concentration 25 mM, 40 mM

or 50 mM) were mixed. The resulting solution was incubated at 37˚C for 60 min. Obtained frag-anti-

gp51 after preparation were used immediately in the further experiments.

2.4. Characterization of the frag-anti-gp51

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Frag-anti-gp51 were characterized by SDS-PAGE analysis [25]. The electrophoresis equipment was

purchased from UVItec (Cambridge, United Kingdom). The Tris-glycine gels were made using an

appropriate concentration of acryl amide (12 %). Protein standards were used as molecular weight

references. The current strength of 10 mA was applied for 5 hours to perform optimal protein

separation. The proteins were stained with Coomassie brilliant blue R-250.

2.5. Preparation of the SPR sensor chip

Before experiment the SPR sensor chip covered with gold (SD AU, XanTec bioanalytics GmbH,

Germany) was cleaned using 0.1 M NaOH solution for 20 min, 0.1 M HCl solution for 5 min. and

piranha solution (1 : 3 = 25 % H2O2 : 75 % H2SO4) for 5 min. Then it was rinsed with pure ethanol

solution and deionized water.

2.6. Immobilization of frag-anti-gp51

The solution containing frag-anti-gp51 was diluted with 0.01 M PBS, pH 7.4, to a final

concentration of 1 mg/mL. A double channel SPR analyzer Autolab ESPRIT obtained from ECO

Chemie (Utrecht, Netherlands) was applied for the monitoring of interactions between the metal

surface and the frag-anti-gp51. A diode laser was used as the light source to produce monochromatic

light with a wavelength of 670 nm. All samples were monitored at a constant of 24˚C temperature.

Immobilization of frag-anti-gp51 was performed in both channels of the SPR cuvette. After the

immobilization the empty sites of the surface were blocked by BSA. For this purpose the SPR sensor

chip modified with frag-anti-gp51 was incubated in 10 mg/mL BSA solution for 12 hours at a room

temperature.

2.7. Immobilization of native anti-gp51

SPR-chip was incubated in 1 mM MUA solution in ethanol at 4 °C for 24 h. Afterward washing

procedure carboxyl groups on the formed MUA self-assembled monolayer were activated with a

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mixture of 0.4 M EDC and 0.1 M NHS in water and functionally active NHS-esters were obtained.

During the next step of experiment immobilization of native anti-gp51 was performed in both

channels of the SPR-cell. Immobilization of 1 mg/mL native anti-gp51 antibodies dissolved in PBS,

pH 7.4, was carried out for 25 min.

2.8. Frag-anti-gp51 interaction with gp51 and the regeneration of the SPR sensor chip surface

The interaction between the immobilized frag-anti-gp51 or native anti-gp51 and gp51 was monitored

after the injection of gp51 solution in PBS, pH 7.4, at corresponding 0.01, 0.025, 0.05, 0.1, 0.2, 0.25

or 0.5 mg/mL concentrations. The interaction was being monitored and followed by a dissociation

step in PBS, 7.4. Then the sensor chip was regenerated with solution consisting of 50 mM NaOH

and 17.34 mM SDS. The regeneration step was carried out for 5 min.

2.9. Calculations

Calibration curves of all investigations were obtained by measuring triplicates and calibration

curve parameters were calculated. The LOD is the lowest quantity of a substance that can be

distinguished from the absence of that substance (a blank value) and was calculated as 3 of the

minimal detectable concentration’s analytical signal. On the other hand, the lowest concentration in a

sample that can be detected with acceptable precision and accuracy means limit of quantification

(LOQ). Precision of created SPR immunosensor in blood serum samples spiked with known

concentration of analyte was evaluated by the determination of the relative standard deviation (RSD).

3. Results and discussion

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3.1. Characterization of frag-anti-gp51 by SDS-PAGE electrophoresis

After the anti-gp51 precipitation native anti-gp51 antibodies were analysed using SDS-PAGE

electrophoresis. The line 3 of figure 1A shows that anti-gp51 antibodies were successfully separated

from other serum proteins and molecular weight is about 160 kDa.

Fig.1.

In order to immobilize biomolecules in oriented way on the SPR-chip coated with gold the

native thiol groups of antibody molecule obtained after reduction might be used. In order to obtain

active antibody fragments with thiol groups, the reduction of anti-gp51 antibodies with DTT (final

concentration 25 mM, 40 mM or 50 mM) or 2-ME (final concentration 15 mM, 35 mM or 70 mM)

was tested. This reaction resulted in the splitting of the S-S bridges between the two heavy chains (in

the hinge region) without affecting the antibody antigen binding sites, i.e. without reduction of

disulfide bounds between the light and heavy chains. The figure 1 illustrates the SDS-PAGE analysis

of the anti-gp51 fragments obtained after reduction with DTT of 50 mM concentration (Fig.1A, line

1) and after reduction with 2-ME of 70 mM concentration (Fig.1B, line 5). Line 1 of fig. 1A, also

line 5 of fig. 1B show one major band around 80 kDa, indicating that a significant amount of frag-

anti-gp51 was produced using both reducing agents. Very similar results (data not shown) were

obtained using lower concentrations of reducing agents, only the major band at 80 kDa was not so

clear. A variety of other previously reported reducing agents (2-mercaptoethylamine) [19] were also

considered using a broad range of experimental conditions [26,27,28,29]. Although the SDS-PAGE

analysis did not prove the best reducing agent at tested concentrations, the frag-anti-gp51 obtained

using DTT (50 mM) or 2-ME (70 mM) were evaluated for the immobilization behaviours and their

antigen binding properties using SPR. Comparative study was done using intact antibodies

immobilized through self-assembled monolayer for the evaluation of antigen binding properties.

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3.2. Comparison of native anti-gp51 and frag-anti-gp51 immobilization methods influence on

antigen binding efficiency

The schematic illustration of stepwise immunosensor design processes of the native anti-gp51 and

frag-anti-gp51 immobilization and selective detection of target using SPR technique is shown in

Fig. 2.

Fig.2.

The native anti-gp51 and frag-anti-gp51 immobilization or native antibody adsorption on the

gold surface was monitored with the SPR (Fig. 3). After the solution of antibodies or their fragments

in the PBS injection into the SPR cell, the SPR angle changes were registered indicating antibody

binding to the chip surface event. The original SPR curves showing the angle shift during

immobilization of different forms of antibodies are presented in Fig.3C. Thus, the SPR angle shifts

for the binding of the frag-anti-gp51 obtained by the reduction with 2-ME and DTT were determined

and compared with other antibody immobilization methods. As shown in Fig. 3A and B, the

reduction of anti-gp51 with different reducing agents influences the antibody immobilization

efficiency and antigen binding capacity.

Fig.3.

The SPR angle shift after the immobilization of frag-anti-gp51 obtained by reduction with

70 mM 2-ME and surface stabilization was 372.4 ± 17.3 m˚ and with 50 mM DDT 348.4 ± 14.1

m˚. The surface density of the frag-anti-gp51 calculated from these angle shifts was 3.05 0.14

ng/mm2 and 2.86 0.11 ng/mm2. The SPR angle shift of 122 m˚ is equivalent to the change in

protein surface density of 1 ng/mm2. The angle shift after native anti-gp51 covalent immobilization

on the self-assembly monolayer and surface stabilization was significantly lower – 73.212.8 m˚ and

the surface density was 0.60.1 ng/mm2 while native anti-gp51 direct adsorption on SPR-chip gold

surface was even lower (0.02 0.01 ng/mm2). Although the adsorption of native anti-gp51 on gold

surface of SPR-chip was registered, after washing procedure with PBS and regeneration solution

almost all antibody molecules were removed from the surface.

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The comparison of the frag-anti-gp51 and full-length native anti-gp51 immobilization

efficiency showed that amount of immobilized molecules is higher for the frag-anti-gp51 obtained

using both reducing agents than for full-length native anti-gp51. However, the highest antigen

binding efficiency (the lowest ratio of immobilized antibody or their fragments and bounded

antigen) was observed using SPR-chip modified with frag-anti-gp51 obtained after reduction with

DTT (Fig. 3B). The native antibodies were attached randomly on self-assembled monolayer which

means that there is a probability of some antibody attachment to the surface surface via their active

sites. The oriented immobilization method is more favourable than already known random

immobilization, because active antibodies fragments have higher antigen binding capacity, the

similar distance between the antigen binding sites and surface, significantly higher antigen

association constants and better stability [19,30,31]. For our further study frag-anti-gp51 obtained

after reduction of anti-gp51with DTT were used.

3.3. Application of frag-anti-gp51 based SPR immunosensor for antigen detection

After the immobilization of frag-anti-gp51 on the SPR-chip surface and subsequent blocking of the

remaining free immobilization sites on the gold layer with BSA in order to avoid non-specific

adsorption of gp51 the surface of SPR-chip was washed and stabilized with PBS, pH 7.4, and

regeneration buffer. When a baseline became steady the solution of gp51 in PBS, pH 7.4, was

injected into the first channel and PBS to the second channel. Interaction of gp51 with the

immobilized frag-anti-gp51 was registered directly (without application of any labels) in real-time,

hence there is no need to designate additional tag molecules (e.g, radioactive, fluorescent). The

formation of frag-anti-gp51/gp51 immune complexes resulted in an increase of the SPR angle. The

SPR angle depends on the refractive index - the environmental situation, in close proximity to the

sensor surface. Frag-anti-gp51 accessibility to the antigen gp51 and immune complex formation on

the surface increased the SPR sensor signal which was recorded.

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In order to reuse the SPR-chip in multiple analyses, the gp51 have to be removed from the

surface, but the immobilized frag-anti-gp51 must remain intact and retain its binding capacity.

Sensor surface regeneration in serial determinations of various analytical samples is an important

part of SPR immunosensors design [32]. The native tertiary structures of different antibodies have

different sensitivity to pH and ionic strength, thus a few regeneration buffers were studied as

potential surface regeneration solutions. After the regeneration step with 0.1 M HCl solution,

removal not only the antigen gp51, but also some changes on the surface modified with frag-anti-

gp51 were observed. These results suggest that 0.1 M HCl affects not only the dissociation of

immune complexes, but also causes the denaturation of frag-anti-gp51 active sites, thus resulting in a

diminished lifetime of the immunosensor. Using regeneration solution consisting of 17.34 mM SDS

and 50 mM NaOH the recovery of the system was observed. The current solution seems to be more

feasible as a regeneration solution for frag-anti-gp51/gp51 complex dissociation and analytical

system for multiple analyses creation. Thus, after the surface regeneration and rinsing with PBS, the

SPR angle attained its original level, which was observed before the injection of the gp51. The

observed changes of the SPR-angle indicated the successful removal of gp51 from the active SPR-

chip surface. Repeated tests can be performed on the same chip using other gp51 concentrations.

In the current research the repeatability of created SPR-based immunosensor was investigated.

The PBS buffer was injected into an SPR-chip until a stable baseline was established. Then,

0.01 mg/mL solution of gp51 was added and SPR signal was recorded for 15 min (Fig. 4). After the

surface regeneration and stabilization procedure, the same concentration of gp51solution was

injected. Repeated detection–regeneration cycles were performed for four times in the same SPR

channel. The injections of the same amount of the gp51 showed almost similar (1-4 %) SPR angle

shifts after 2nd and 3rd interaction with antigen. These results clearly indicate that the frag-anti-gp51

modified surface retained its reactivity almost completely during the three detection–regeneration

cycles of the SPR-chip. However, after 4th cycle the SPR angle shift was lower by 15 %. Tsai et al.

reported even higher decrees of SPR angle shift after 3 detection-regeneration cycles and it was

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about 30% [9]. The application of immunosensors for multiple analyses makes analytical procedures

more cost-efficient and in particular cases it reduces the duration of the analysis.

Fig.4.

In order to investigate the sensitivity of the frag-anti-gp51 based immunosensor samples with

various concentrations of gp51 were prepared. Figure 5A shows SPR sensograms for six different

concentrations of gp51 ranging from 0.01 to 0.5 mg/mL. The SPR-signal after establishment of

steady-state conditions (at “the equilibrium angle”) was plotted against the concentrations of gp51

(Fig. 5B). As a result the frag-anti-gp51/gp51 complexes formation on the sensor surface was

depend on the gp51 concentration in the samples (Fig. 5B).

Fig. 5.

Within the examined antigen gp51 concentration range (0.01 0.5 mg/mL) a linear

dependence (R2=0.9974) was observed only in the concentration interval from 0.01 to 0.1 mg/mL. It

was demonstrated that for the developed SPR-immunosensor the LOD is 0.0028 mg/mL and LOQ –

0.0092 mg/ml, respectively. Generally, the dependence of equilibrium angle upon all tested

concentrations of antigen is not linear as it is presented in Fig. 5B.

During earlier experiments of our group [31] the conventional Langmuir kinetic equation was

applied for the determination of affinity constants after measurements of gp51 interaction with

native anti-gp51 and frag-anti-gp51 by total internal reflection ellipsometry. The same nonlinear

tendency was observed in current study. The SPR based method enables direct determination of

binding constants for a variety of specific antigen-antibody interactions in real-time [10].

For the calculation of BLV antigen and frag-anti-gp51 antibody complex formation

efficiency the equation was used [9]:

%100

Ag

Ab

Ab

Ag

M

M

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where: MAb – molecular mass of antibody fragments, ΔΘAg – SPR angle change after antigen

binding; MAg – molecular mass of gp51; ΔΘAb – SPR angle change after frag-anti-gp51

immobilization.

Taking into account the BLV gp51 antigen molecular mass of 51 kDa [33] and frag-anti-gp51

molecular mass of 80 kDa, the complex formation efficiency of 84.4% was calculated. Other authors

by SPR immunosensor for staphylococcal enterotoxin B [9] observed that in the case of the random

antibody orientation the antigen binding efficiency was in the rage of 60 %, while oriented antibody

immobilization results in a 90 % of antigen binding efficiency.

3.4. Detection of gp51 in real samples by SPR-based immunoassay

The feasibility of immunosensor to detect gp51 under optimized conditions using SPR device was

investigated in spiked blood serum samples. Recoveries of the proposed method were in the rage of

85 – 100 % and are presented in Table 1. It was found that better recovery was achieved by

increasing concentration of analyte. The results of precision expressed as RSD % values were

calculated for different antigen concentrations from 0.01 to 0.2 mg mL-1.

4. Conclusions

The effect of different reduction agents on the frag-anti-gp51 production and immobilization on

SPR-chip, also on gp51 binding capacity was studied and compared with system where random

native antibody immobilization was applied. The best gp51 detection efficiency was monitored using

SPR-chip modified with oriented antibody fragments obtained after the reduction with dithiothreitol

at 50 mM concentration. The developed SPR sensor offered the LOD as low as 0.0028 mg/mL while

the LOQ was as low as 0.0092 mg/mL with very good repeatability (in the range of 1-4 %) during

the three detection–regeneration cycles. The detection of antigen with surface modified by

antibodies takes about 4-15 min and it depends on the concentration of analyte in the tested sample.

Immobilization of frag-anti-gp51 was found to be more suitable for the design of SPR-

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immunosensor for gp51 detection if compared to random immobilization of anti-gp51. In

comparison to other methods used for the identification of animals infected with bovine leukemia

virus, e.g., enzyme-linked immunosorbent assay, proposed immunosensor is simpler to handle, it is

much faster analytical tool suitable for the application in medical diagnosis, and can be used for

multiple analyses. This method was successfully applied for the detection of bovine leukemia virus

antigen gp51 in blood serum in a rapid, reliable and selective manner.

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Biographies

Julija Baniukevic is a Ph.D. student and received her master degree in 2009 with specialization in bioengineering. She is doing her doctoral research in Vilnius University, Faculty of Chemistry, Research Center for Nanotechnology and Materials Science-NanoTechnas. Julija focused her research on design of optical immunosensors. She takes part in FP7 project „NanOpinion“ on monitoring public opinion on Nanotechnology in Europe.

Justina Kirlyte is a Ph.D. student and received her master degree in 2009 with specialization in chemistry. She is doing her doctoral research in Vilnius University Faculty of Chemistry, Research Center for Nanotechnology and Materials Science-NanoTechnas. Her research areas mostly refer to biosensors creation of human growth protein.

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Prof. dr. Arunas Ramanavicius is a professor at Vilnius University, Vilnius, Lithuania. He is head of Department of Physical Chemistry at Vilnius University and Research Center for Nanotechnology and Materials Science-NanoTechnas. In 1998 he received his PhD degree and in 2002 he received doctor habilitus degree from Vilnius University. He is serving as expert-evaluator in EU-FP7 program coordinated by European Commission and he is technical advisor to many foundations located in European and non-European countries. He has research interests in bionanotechnology, nanomaterials, biosensorics, bioelectronics, biofuel cells and MEMS based analytical devices. Assoc. prof. dr. Almira Ramanaviciene. She received her PhD degree in biomedicine in 2002 from the Institute of Immunology and Vilnius University. In 2008 she completed habilitation procedure in Physical Sciences at Vilnius University. She is serving as FP7 projects expert for the European Commission and other international foundations. She has research interests in the field of nanotechnology, immunoassays, conducting polymers, bio- and immuno-sensors and MEMS based analytical devices.

Legends to figures:

Fig.1. SDS-PAGE analysis (12 % gel; non-reducing conditions) of purified native anti-gp51

antibodies before (A, line 3) and after (A, line 1 and B, line 5) reduction. A - frag-anti-gp51 obtained

after reduction with DTT (50 mM) (line 1), protein molecular weight marker (line 2) and purified anti-

gp51 (line 3); B - protein marker (line 4) and frag-anti-gp51 obtained after reduction with 2-ME

(70 mM) (line 5).

Fig. 2. Schematic illustration of SPR-based immunosensors for gp51 detection using different

antibody immobilization on gold surface of SPR-chip methods. A – Immunosensor based on

randomly oriented antibodies covalently immobilized on self-assembled monolayer. B -

Immunosensor based on randomly adsorbed native antibodies on gold surface. C - Immunosensor

based on oriented immobilized fragmented antibodies (frag-gp51).

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Fig.3. A - Comparison of the surface densities after antibody or their fragments immobilization on the

SPR-chip (green bars) and after interaction with antigen (red bars). 1 – Frag-anti-gp51 obtained after

reduction with 2-ME were oriented immobilized; 2 - frag-anti-gp51 obtained after reduction with DTT

were oriented immobilized; 3 - native full-length anti-gp51 were randomly immobilized through self-

assembled monolayer and 4 - native full-length anti-gp51 were randomly adsorbed on the on the SPR-

chip. B - The ratio of the amounts of immobilized antibody or their fragments and bounded antigen. C

- The original SPR curves showing the angle shift during immobilization of different forms of

antibodies.

Fig. 4. A - The SPR sensor response recovery using 0.01 mg/mL solution of gp51. B - The original

SPR curve registered during the recovery test.

Fig. 5. A - The SPR sensorgrams using different concentrations of gp51. (a – 0.5 mg/mL; b – 0.25

mg/mL; c – 0.1 mg/mL; d – 0.05 mg/mL; e – 0.025 mg/mL; f – 0.01 mg/mL). Numbers show the

stage of kinetic curves (1 – baseline, 2 – association, 3 – dissociation, 4 – regeneration, 5 – return to

baseline).

Table 1. Recovery of gp51 in blood serum samples using SPR-chip modified with oriented antibody

fragments.

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kDa

116

66.2

45

35

25

18.4

14.4

A

1 2 3

kDa116

66.2

45

35

25

18.4

14.4

4 5

B

Fig. 1.

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Fig. 2.

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Antibodies immobilization methods1234Surface density, ng/mm201234Ab immobilization on Au surfaceInteraction with Ag AAntibodies immobilization methods01234Ratio1234567BTime, s050010001500SPR angle shift, mo02004006008001 2 4 3 C

Fig. 3.

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Time, s0 500 1000 1500

SP

R a

ng

le s

hif

t, %

0

20

40

60

80

100

Time, s

0 200 400 600 800 1000

SP

R s

ign

al r

eco

very

, %

0

20

40

60

80

100

1st, 100%2nd, 99%3rd, 96%4th, 85%

A B

Fig. 4.

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Time, s

0 500 1000 1500

SP

R a

ng

le s

hif

t, m

o

0

20

40

60

80

100

120

140

160

180 a

1

2

3

4 5

b

c

d

e

f

Concentration, mg/mL

0,0 0,1 0,2 0,3 0,4 0,5

Eq

uil

ibri

um

an

gle

sh

ift,

mo

0

20

40

60

80

100

120

140A B

Fig. 5.

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Table 1.

Added concentration of

gp51, mg/mL

Detected concentration

of gp51, mg/mL

Recovery ratio,

%

RSD,

%

0.01

0.025

0.008

0.017

80

85

9.21

9.05

0.05 0.046 92 8.96

0.1 0.1 100 8.67

0.2 0.2 100 8.45