Beruflich Dokumente
Kultur Dokumente
Received 30 March 2006; received in revised form 4 August 2006; accepted 9 August 2006
Available online 7 September 2006
Abstract
This work describes the development of an electrochemical, recombinant Fab fragment-based immunosensor for the detection of testosterone
in bovine urine. The sensor comprised of a testosterone conjugate on the surface of screen-printed electrodes, and recognition followed by an
anti-testosterone Fab fragment. The use of an IgG-horseradish peroxidase conjugate determined the degree of competition. Chronoamperometry at a
potential of +100 mV, was chosen to reductively measure the product of the catalysis of 3,3 ,5,5 -tetramethylbenzidine catalysis. ELISA was primarily
used to investigate the assay system, prior to transferring to SPEs. The final Fab-based sensor exhibited the linear range of 300–40,000 pg/ml with
limit of detection of 90 ± 13 pg/ml. Furthermore, the developed Fab sensor allowed for the determination of testosterone in bovine urine directly
after dilution, omitting the necessity of extraction and hydrolysis. Comparison of administrated bovine urine samples between the developed Fab
sensor and GC–MS data showed quantitative or semi-quantitative results and enabled identification of suspicious samples for further extensive
analysis by established analytical techniques. With simple sample preparation, low limit of detection, and good repeatability, the proposed method
can offer alternative advantages as a primary screening tool for meat quality control.
© 2006 Elsevier B.V. All rights reserved.
1. Introduction and suffer from considerable time delays between sampling and
obtaining results. These disadvantages limit their routine use and
Testosterone is an anabolic steroid often used for illegal restrict assaying to extensive laboratory environments.
growth promotion in livestock farming and thereby causing Immunological techniques are based on the molecular recog-
potential health risks to consumers. The use of steroids for nition of antigens by antibodies to form a stable complex which
growth promoting purposes in animals is banned in the Euro- are capable of detecting low level concentrations of analytes in
pean Union for food safety reasons (Directive 88/146/EEC). The biological matrices. Various assays have been developed to mea-
determination of residues is required in animals and in fresh meat sure testosterone in plasma (Rajkowski et al., 1989), serum (Dhar
as given by the EU Directive (86/849/EEC). and Ali, 1992), and human urine (Al-Dujaili, 2006). The cumber-
There are various analytical methods used for the detection of some handling procedures involved, hinder their use as on-site
testosterone in biological fluids including, HPLC (Li et al., 2002; detection systems. By comparison, immunosensors combining
Navajas et al., 1995), GC–MS (Samuels et al., 1998; Saudan et sensitivity of the antibody–antigen interaction with other advan-
al., 2004) and LC–MS offers a simplified, specific and sensitive tages, including cost-efficient, speed of analysis and portable
alternative to GC–MS (Leinonen et al., 2004). Whilst these tech- screening, allows for fast, decentralised measurement away from
niques have been used as quantitative and confirmatory methods, the lab environment. They are widely applied in the area of
they do need expensive instrumentation, specialized personnel human and animal health and in the food sector (D’Orazio, 2003;
Mello and Kubota, 2002). Electrochemical-based screen-printed
electrodes (SPE) provide an approach to develop cost-effective
∗ Corresponding author. Tel.: +34 93 553 40 32; fax: +34 93 553 40 00. devices through disposable means and have been reported to
E-mail address: mark.kreuzer@icfo.es (M.P. Kreuzer). measure steroids, like progesterone in cow’s milk (Pemberton
0956-5663/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2006.08.002
H. Lu et al. / Biosensors and Bioelectronics 22 (2007) 1756–1763 1757
et al., 1998; Kreuzer et al., 2004) and estradiol in human serum immobilised on microtiter plate wells. For large-scale, soluble
(Pemberton et al., 2005). production of Fab 220, the Fab expression unit with six histidine
Current immunoassays capable of detecting steroid hor- residues at the C-terminus of the heavy chain was cloned into
mones are still performed using polyclonal and monoclonal the pKKtac expression vector (Takkinen et al., 1991). The Fab
antibodies. It has been suggested that the main limitation of 220 was produced in E. coli strain RV308 (ATCC 31608) by
immunosensor technology is related to the antibodies and their high cell density fermentation and purified by immunoaffinity
properties (Hock, 1997). Steroid hormones are small, rigid, and protein G chromatography as described in Nevanen et al.
hydrophobic molecules with only a few functional groups capa- (2001).
ble of specific interaction with antibodies. Furthermore, they
are poorly immunogenic in mice and rats; two species from 2.2. Apparatus
which monoclonal antibodies are usually generated, making it
difficult and expensive to produce. The supply of good poly- Ninety-six-well flat-bottomed polystyrene microtiter plates
clonal antibodies of uniform quality is not easy and requires were purchased from NUNC (Dublin, Ireland). Bio-Tek Instru-
continuous immunization of many laboratory animals (Killard ments (VT, USA) supplied the microplate reader (model EL
et al., 1995). Antibody engineering provides excellent tools to 311). Incubations at elevated temperatures were carried out in
tailor the properties of antibodies in respect to affinity, speci- a thermostat oven supplied by Heraeus Instruments (Hanau,
ficity and performance for different applications. The possibility Germany). Electrodes were prepared using a DEK (model
to express soluble, active antibody fragments, Fab and scFv, 247) screen-printer (Dorset, UK). Chronoamperometric mea-
in E. coli (Better et al., 1988; Bird et al., 1988; Huston et al., surements were performed using a potentiostat (AUTOLAB)
1988) allows cost-effective production, feasible engineering of with GPES software (ECO-CHEMIE, Netherlands).
the binding properties and fusions with peptide tags enhanc-
ing purification, detection and immobilisation. Furthermore, the 2.3. Urine sample preparation
antibody phage display library technology has opened com-
pletely new possibilities to produce novel binding specificities Urine samples of heifers injected intramuscularly with testos-
avoiding the limitations inherent in the mammalian immune terone (200 mg per animal) were provided by Dr. M. Crowe
response, as reviewed in Hoogenboom (2005). A number of (Faculty of Veterinary Medicine, National University of Ire-
recent publications describe the development of recombinant land, Dublin). The study was performed in compliance with
antibody fragments to steroid analytics by antibody engineer- protocols approved by the Ethics Committee, University Col-
ing techniques (Hemminki et al., 1998; Valjakka et al., 2002). lege Dublin, the Cruelty to Animals Act (Ireland, 1876), and
To our knowledge, there is little information of the application the European Union Directive 86/609/EC. An aliquot (1 ml) of
of a recombinant Fab fragment to detection of testosterone in the urine specimen of interest was defrosted and centrifuged at
electrochemical immunosensor area. 3000 × g for 3 min. The supernatant was collected, aliquoted
(20 l) and frozen at −20 ◦ C until assayed to avoid repeated
2. Materials and methods freeze–thaw cycles of samples.
Testosterone, 19-nortestosterone, boldenone, methyltestos- Ninety-six-well ELISA plates were coated with 50 l of the
terone, methylboldenone, eticholanolone, epieticholanolone, appropriate dilution of testosterone–BSA conjugate in phos-
epitestosterone, 4-androsten-3,17-diol, 4-androsten-3␣,17- phate buffered saline (PBS, pH 7.4) at 37 ◦ C for 45 min. After
diol, 2␣-, 6-, 16␣-, and 16-hydroxytestosterone were washing three times with washing buffer (PBS containing 0.05%
purchased from Steraloids Inc. (RI, USA). Bovine serum (v/v) Tween 20), 200 l per well of blocking buffer (PBS con-
albumin (BSA), testosterone 3-(o-carboxymethyl)oxime: BSA taining 1% (w/v) BSA) was added and plates were then incu-
conjugate, anti-mouse IgG peroxide conjugate, 3,3 ,5,5 - bated at 37 ◦ C for 45 min or at 4 ◦ C overnight followed by wash-
tetramethylbenzidine dihydrochloride (TMB) were purchased ing as described above. Thereafter, an optimised, one-step incu-
from Sigma–Aldrich (Dublin, Ireland). The inks used in screen- bation protocol was used for the remaining immunoreagents. A
printing (Electrodag® B-0851, 423-SS and 451-SS) were pur- volume of 25 l of each different concentration (0–1000 ng/ml)
chase from Acheson (Plymouth, UK). All other solvents and of testosterone standards, 25 l of the suitable dilution of the Fab
reagents were of analytical grade and were purchased from fragment and 25 l enzyme labelled anti-species (␣-IgG–HRP)
Sigma–Aldrich (Dublin, Ireland). were all added at the same time into the wells and incubated at
The anti-testosterone Fab fragment (clone 220) was iso- 37 ◦ C for 15 min. After a further washing step, 100 l per well
lated from a phage display library constructed from mice of substrate solution containing 0.1 mg/ml TMB and 0.2% (v/v)
immunized with testosterone 3-(o-carboxymethyl)oxime: BSA hydrogen peroxide in 0.05 M phosphate citrate buffer (pH 5,
(Sigma–Aldrich). The phage library construction, selection and containing 0.1 M potassium chloride) were added and the wells,
clone analysis was essentially done as described in Pulli et al. left to develop colour for 30 min at room temperature and subse-
(2005). The Fab clone 220 was isolated by selecting the library quently stopped by the addition of 50 l of 2 M sulphuric acid.
for four rounds with the immunogen, testosterone-CMO-BSA, Absorbance was read at 450 nm. Standard-dose response curves
1758 H. Lu et al. / Biosensors and Bioelectronics 22 (2007) 1756–1763
were constructed by plotting the absorbance as a function of ELISA was primarily performed in 96-well plates to investigate
testosterone concentration. assay performance due to its simple and quick determination of
protein characteristics.
2.5. Electrochemical immunosensor protocol
3.1.1. Optimisation of ELISA parameters
The electrode design and layer development were prepared Optimum working concentrations of both coating conju-
in advance by the systematic and controlled deposition of the gate and anti-testosterone Fab fragment were obtained by
varying layers (conducting, isolating and working) onto a PVC performing chessboard titrations, whereby serial dilutions of
sheet. After printing the last path, the electrodes were cured at both variables were carried out in duplicate (data not shown).
90 ◦ C for 1 h. The electrodes consisted of a silver conducting Optimum concentrations of coating testosterone–BSA conju-
ink coated by an insulating layer and a carbon working area of gate was found to be 2.5 g/ml with recombinant Fab frag-
0.16 cm2 . Immunoassays were performed on this working area. ment around 0.31 g/ml corresponding to 80% of the maxi-
The SPE were cut from the PVC sheet into 45 mm × 15 mm mum signal. Subsequent ELISA assays were carried out under
long strips. The electrodes were modified as follows: a 5 l these conditions incorporating a 1/600 dilution of IgG–HRP.
drop of appropriately diluted coating testosterone–BSA conju- This was found to catalyse the turnover of substrate to prod-
gate in PBS buffer was spread onto the surface of the working uct efficiently and within a short time suitable for ELISA
electrode and incubated for 30 min at 37 ◦ C. The electrodes measurements.
were then washed with washing buffer followed by distilled
water to remove unbound coating conjugate and excess water 3.1.2. Competitive immunoassay using ELISA
was allowed to evaporate at room temperature. The electrode The competition study was carried out setting up two proto-
surface was then blocked by the addition of 100 l blocking cols: in the first protocol (one step), analyte, Fab and IgG–HRP
buffer and incubated for 45 min at 37 ◦ C. For the competition were mixed together directly in the microtiter well where the
step, the electrodes were immersed in 10 l of a 1:1 mixture coating conjugate had been immobilised. In the second one (two
of analyte and suitable dilution of Fab fragment. For urine steps), analyte and Fab fragment were added into the microtiter
analysis, a frozen aliquot was defrosted and diluted in PBS wells followed by the addition of IgG–HRP as a subsequent step.
(1/10, v/v). Dilutions of testosterone were then made with this The competitive curves showed there was no difference between
modified urine stock. After incubation and a washing step, two different protocols. Thus, the first protocol was used in
the electrodes were covered with 10 l IgG–HRP conjugate subsequent assays because shorter assay time could be used.
and incubation for 30 min at 37 ◦ C followed. The electrodes Different incubation times (15, 30, 45, 60 min) of the competi-
were washed for a final time with distilled water and mea- tion one-step were studied and the optimum time was determined
sured using chronoamperometry at +100 mV by adding TMB as 15 min at 37 ◦ C.
substrate solution (0.24 mM TMB in phosphate citrate buffer, A competitive assay was established between immobilised
pH 5, with 1.32 mM H2 O2 ) over all three electrodes (work- testosterone–BSA conjugate and analyte for Fab fragment.
ing, reference and auxiliary electrodes) in a non-hydrodynamic Blank wells and controls were incorporated for measurement
manner. of maximum signal (zero analyte) and for non-specific binding
All electrochemical experiments were performed at room (zero Fab). Fig. 2a shows averaged standard curves by ELISA.
temperature. The parameters for chronoamperometry detec- Data is presented as the average of nine separate assays while
tion were as follows—pre-treatment: first conditioning potential within each assay, each data point was the average of triplicate
200 mV, duration 20 s, equilibrium time 5 s; measurement: inter- measurements (n = 3, N = 9). Calibration curve statistics are pre-
val time 0.1 s, standby potential 200 mV, number of potential sented in Table 1. Repeated analysis confirmed these findings.
steps 1; potential 100 mV; duration 10 s. All data points were At this point it was evident that the developed recombinant Fab
carried out in duplicate and each value was the mean value of could be used as a recognition element, with suitable sensitivity
two readings. The measured current was recorded and plotted for the development of the electrochemical sensor and no further
against testosterone concentration to give a calibration plot. Cali- effort was placed on the ELISA system. Complete optimisation
bration curves were freshly prepared prior to each assay by using and analysis would be performed using the screen-printed elec-
10 serial dilutions from standard solutions with concentrations trode set-up.
in the range of 0–1000 ng/ml.
Table 1
3. Results and discussion Best-fit parameters for repeated analysis for testosterone using screen-printed
electrodes and ELISA
3.2. Fab-based immunosensor using screen-printed the results obtained with the different concentrations of proteins
electrodes and chronoamperometric detection (coating and Fab fragment). The effect of increasing the coating
testosterone–BSA conjugate and Fab fragment (Fig. 1a) was
In biological fluids, ascorbic acid, uric acid and xanthine to increase the overall signal. However, higher concentrations
are common electrochemical interferences. In a previous study did not yield significantly larger signals and above 20 g/ml of
(Jamal et al., 2005), ascorbic acid was shown to be the major testosterone–BSA, no increase in current was observed. Gener-
electrochemical interference using p-aminophenol as a model ally speaking, as a starting point, we tend to use a combination of
system. Chronoamperometry was the most sensitive method reagents that attains a current of ca. 1 A. This yields a sufficient
among a range of electrochemical techniques tested (differ- sensitivity and spread of data points from a maximum signal of
ential pulse voltammetry, square wave voltammetry, linear 1 A to a background typically found at 10–15% or 150 nA. If
sweep voltammetry, chronoamperometry) in the detection of sensitivity is subsequently a problem, dilutions of reagents can
p-aminophenol on a glassy carbon electrode. Therefore, the be adjusted to alter the precision, sensitivity and quantitative
electrochemical technique chosen in this study was chronoam- range of the assay (Ekin, 1993). Theory indicates that decreas-
perometry performed at a potential of +100 mV where the TMB ing concentrations of antibody will afford greater sensitivity as it
product undergoes reduction. The SPEs are produced in batches will require less analyte to reach a constant reference level, such
of 30. Within a batch the typical error recorded for measuring as the EC50 . This approach, however, is limited by the precision
standard redox reactions was about 1% or less. Once the SPEs achieved with increasing dilution (Ruth, 2001). This theory was
have bioreagents immobilised, error accounts for ca. 8 ± 2% applied to Fab fragment and from Fig. 1b it can be seen that lower
depending on both the handling/manipulation (operator error) Fab fragment concentrations shift the standard curves to the
and also the sample matrix. This value is quoted for buffered left, decreasing the limit of detection and the EC50 favourably.
systems. When more complex matrices are used, error is gener- Cost may also be a factor in favour of using lower concentra-
ally higher with errors between 10 and 15% noted. tions of proteins. But also this approach decreased the absolute
signal and thus the Hillslope, an important parameter when
3.2.1. Optimisation of electrochemical parameters determining assay sensitivity and detection. Therefore optimum
Due to the differences in surface area of the screen-printed compromised concentration of coating testosterone–BSA con-
electrode (0.16 cm2 ) compared to the conventional microtiter jugate was found to be 20 g/ml with recombinant Fab fragment
plate (0.28 cm2 ) and the binding capacity of both surfaces, of 0.5 g/ml.
optimisation of reagents was re-evaluated. As for the ELISA Different incubation time (15, 30, 45, 60 min) of coating con-
study, assay parameters such as the amount of proteins (coat- jugate, Fab fragment and enzyme labelled anti-species on SPE
ing testosterone–BSA conjugate and Fab fragment) and enzyme were also studied and optimum incubation time for each step
labelled anti-species, incubation time of each step were re- was found to be 30, 45 and 30 min at 37 ◦ C, respectively (data
evaluated and optimised again. not shown).
Testosterone–BSA is passively adsorbed to the surface
of the SPE. From Fig. 1a, the currents noted when using 3.2.2. Competitive immunoassay on screen-printed
testosterone–BSA coating concentrations at 5, 10, 20 or electrodes
40 g/ml were obviously higher than 0 g/ml testosterone–BSA A competition study using one-step and two-step incubation
conjugate coating (non-specific binding control). In the latter protocols was also investigated for the electrochemical system
case, the SPEs were subsequently blocked in PBS buffer (1% and the results showed that competitive one-step incubation did
BSA), Fab fragment added at various concentrations and the sig- not give as high signal as two-step incubation as in ELISA case
nals determined that an average of ca. 300 nA was attributed to previously determined. This difference presumably reflects the
background and/or non-specific signal. Fig. 1a and b represents relatively high protein-binding capacity of commercially treated
Fig. 1. (a) Optimisation of coating testosterone–BSA conjugate and Fab concentrations in the presence IgG–HRP (1/500) on SPEs (n = 2). Coating concentration
(g/ml): , 40; , 20; , 10; ×, 5; *, 0. (b) Competitive immunoassay on SPEs using different concentrations of Fab (g/ml): , 4; , 1; 䊉, 0.5; , 0.25 (n = 2).
Fixed amount of coating testosterone–BSA conjugate (20 g/ml) and IgG–HRP (1/500 dilution) were used. Chronoamperometric detection at +100 mV.
1760 H. Lu et al. / Biosensors and Bioelectronics 22 (2007) 1756–1763
Fig. 2. Competitive assays for testosterone detection using Fab fragment as the molecular recognition element (a) ELISA data (n = 3, N = 9) and (b) chronoamperometric
detection at +100 mV using SPEs (n = 2, N = 3). Maximum value represents zero analyte (testosterone).
microtiter plates compared to the carbon working surface of the In order to measure the non-specific binding of the secondary
SPEs. Thus, two incubation steps were used for the electrochem- label (at various dilutions) to testosterone–BSA coating conju-
ical system. gate (20 g/ml) we noted a specific current of 900 nA and a
Fig. 2b shows competitive immunoassay curve for testos- non-specific one of 143 nA (specific:non-specific ratio of 6.3)
terone detection on SPEs using the optimised parameters found for a 1:500 dilution. Background increased significantly when
previously. It can be seen that on decreasing the analyte con- lower dilutions of secondary label were used (710 nA) while
centration, the signal approaches the maximum control for the decreasing the specific:non-specific ratio to 4.7.
competitive immunoassay. For this assay, the linear range can The sensitivity of the Fab sensor was determined by both
be observed between 300 and 40,000 pg/ml. The list of param- EC50 and LOD parameters. To calculate LOD, the following
eters for repeated analysis for testosterone on SPEs, which parameters of EC50 , Hillslope and three times of standard devi-
were also obtained in the ELISA studies are summarized in ation of the maximum signal were used and values returned
Table 1. It was found that the EC50 parameter was very close to were based on the average of three separate assays in triplicate
that of the ELISA system. Regression values (R2 ) of the elec- measurements. The Hillslope, which gives a measure of the sen-
trochemical response curve showed that accuracy was good, sitivity of the assay, is measured by a tangent through the point
while error (average R.S.D. across all points of curve) was of inflection of the sigmoid (EC50 ). Values of Hillslope close
still acceptable (10%). This can be attributed to the exces- to 1 are considered optimum because of the inverse proportion-
sive handling of the electrodes and the inability at present to ality of the signal to concentration. Determination of limit of
measure large numbers of electrodes simultaneously. These val- detection also uses this parameter and thus is an important char-
ues are thought acceptable and showed the robustness of the acteristic of any curve as a deviation from a value of 1 attributes
assays. to a less sensitive assay based on the following equation:
Non-specific binding of the sensor was determined in a num-
−1/k
ber of ways. In Section 3.2.1, different Fab fragment concentra- Max − Min
tions (0, 0.032, 0.16, 0.8, 4 and 20 g/ml) at different coating LOD = EC50 −1
Max − Min − 3S.D.max
concentrations was compared (Fig. 1a). The current noted when
the Fab fragment concentration was zero was proportional to the where EC50 is an effective concentration for 50% inhibition;
non-specific binding of the secondary label (IgG–HRP; 1:500 Max and Min are maximum and minimum signal; S.D.max is
dilution). When the optimum protein conditions were subse- standard deviation of maximum signal; k is Hillslope. The LOD
quently used to give the competitive assay (Fig. 2b), the non- was found to be 90 ± 13 pg/ml based on this equation. Over
specific binding or background was noted in the plateau at high the past decade, numerous publications concerning testosterone
testosterone levels (>100 ng/ml). This value was recorded as ca. immunoassay measurement using various whole antibodies as
150 nA. the molecular recognition element have been reported (Table 2).
Table 2
Immunoassays for testosterone detection (from literature) with associated limits of detection
Antibody used Medium LOD (pg/ml) Reference
Rabbit antiserum against testosterone 3CMO-BSA Human urine NA Hanquez et al. (1987)
Rabbit antiserum against testosterone 3CMO-BSA Plasma 60–2000 Rajkowski et al. (1989)
Rabbit antiserum against testosterone 3CMO-BSA Serum 100 Dhar and Ali (1992)
Rabbit antiserum against testosterone 3CMO-BSA Culture medium 11.43 Illera et al. (1997)
Rabbit antiserum against testosterone 3CMO-BSA Human serum 15 Shrivastav et al. (2003)
␣-Testosterone antibody raised in sheep (Micropharm, London, UK) Buffer 12.2 Al-Dujaili (2006)
H. Lu et al. / Biosensors and Bioelectronics 22 (2007) 1756–1763 1761
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M., Söderlund, H., Teeri, T.T., 2001. J. Chromatogr. A 925, 89–97. 1995. J. Chromatogr. B 672, 207–215.
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