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Ochratoxin A in Some French

Wines: Application of a Direct
Competitive ELISA Based on an
OTA–HRP Conjugate
a a b a
A. Radoi , L. Dumitru , L. Barthelmebs & J.-L.
a
Marty
a
IMAGES, Université de Perpignan , Perpignan ,
France
b
Facultatea de Stiinta si Ingineria Materialelor,
Catedra de Chimie, Universitatea Transilvania
Brasov , Brasov , Romania
Published online: 28 May 2009.

To cite this article: A. Radoi , L. Dumitru , L. Barthelmebs & J.-L. Marty (2009)
Ochratoxin A in Some French Wines: Application of a Direct Competitive ELISA
Based on an OTA–HRP Conjugate, Analytical Letters, 42:8, 1187-1202, DOI:
10.1080/00032710902890447

To link to this article: http://dx.doi.org/10.1080/00032710902890447

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 Analytical Letters, 42: 1187–1202, 2009
Copyright # Taylor & Francis Group, LLC
ISSN: 0003-2719 print=1532-236X online
DOI: 10.1080/00032710902890447

IMMUNOASSAY

Ochratoxin A in Some French Wines: Application

of a Direct Competitive ELISA Based
Downloaded by [Dicle University] at 06:57 11 November 2014

on an OTA–HRP Conjugate

A. Radoi,1 L. Dumitru,1,2 L. Barthelmebs,1 and J.-L. Marty1

1
IMAGES, Université de Perpignan, Perpignan, France
2
Facultatea de Stiinta si Ingineria Materialelor, Catedra de Chimie,
Universitatea Transilvania Brasov, Brasov, Romania

Abstract: In the direct-competitive enzyme-linked immunosorbent assay

(dc-ELISA) approach, an ochratoxin A–horseradish peroxidase (OTA–HRP)
synthesized conjugate was utilized. Fluorescence studies revealed that when
excited at 333 nm, the OTA–HRP conjugate shifted in fluorescence from
438 nm to 444 nm. At the 295-nm excitation wavelength, the tryptophan emission
peak blue-shifted from 326 nm to 318 nm. Western blot analysis revealed that
anti-OTA antibodies specifically binds and recognize the OTA–HRP conjugate.
The dc-ELISA showed an IC50 value of 400 ng=L and a linear working range
(LWR) value between 100 and 1500 ng=L. The ELISA results were confirmed
with high-performance liquid-chromatography (HPLC) analysis to assess the
potential of the OTA–HRP conjugate.

Keywords: ELISA, fluorescence, HPLC, OTA, Western blot, wine

Received 2 December 2008; accepted 9 March 2009.

Address correspondence to A. Radoi, IMAGES, Université de Perpignan, 52
Avenue Paul Alduy, 66860 Perpignan Cedex, France. E-mail: radoiantonio@
yahoo.com

1187
 1188 A. Radoi et al.

INTRODUCTION

Mycotoxins are natural, secondary metabolites produced by different

microfungi (Aspergillus, Penicillium, and Fusarium species) on agricul-
tural commodities, and in humans or animals chronic exposure is capable
of causing cancer, kidney toxicity, immune suppression, and even death
(Bennett and Klich 2003). Ochratoxine A (OTA) is the most common
naturally occurring mycotoxin and is produced mainly by Aspergillus
ochraceus, Aspergillus carbonarius, and Penicillium verrucosum (FAO=
WHO 2001). It occurs on a wide variety of cereals (wheat, barley, maize,
Downloaded by [Dicle University] at 06:57 11 November 2014

oats), dried fruits, spices, and coffee and in fermented beverages (beer and
wine), even in milk (IARC 1993; Battaglia, Hatzold, and Kroes 1996;
Trucksess et al. 1999; Jorgensen 1998). Because of its widespread occur-
rence on such a large variety of agricultural commodities and because
of the potential health risks, mainly toward humans, OTA has been clas-
sified by different international and national organizations as a possible
carcinogen to humans (WHO 1996), as a nephrotoxic agent (Castegnaro
et al. 1991), and as an immunosuppressor (IARC 1993). In 2001, JECFA
(Joint FAO=WHO Expert Committee on Food Additives) set a provi-
sional tolerable weekly intake (PTWI) of 100 ng kg
1 b.w., corresponding
to approximately 14 ng kg
1 b.w. per day (FAO=WHO 2001).
Recent studies have shown that in the European diet, wine has
been identified as the second major source of human exposure to OTA,
following cereals (Serra et al. 2003). The European Commission, through
Commission Regulation (EC) No. 123=2005 of 26 January 2005, which
amended Regulation (EC) No. 466=2001 regarding OTA, set the maxi-
mum levels of OTA in wine (red, white, and rosé) and other wine- and
grape-based beverages at 2 mg=kg or ppb. The same regulation fixes the
maximum levels for raw cereal grain at 5 mg=kg, for soluble coffee at
10 mg=kg, and for baby foods and processed cereal-based foods for
infants and young children at 0.50 mg=kg.
In most cases, the detection of OTA involves instrumental techni-
ques, such as liquid chromatography (LC) coupled with fluorescence
(MacDonald et al. 1999; Vatinno et al. 2008; Meletis, Meniades-
Meimaroglou, and Markaki 2007), mass detection (Lau et al. 2000;
Goryacheva et al. 2006; Timperio et al. 2006), and=or capillary-zone
electrophoresis with laser-induced fluorescence (Corneli and Maragos
1998; Köller et al. 2004). In recent years, however, novel technologies
combined with immunochemical assays have been proposed for rapid
quantitative or semiquantitative analysis of OTA in food and beverages.
They include lateral flow devices (Danks et al. 2003), flow-through
enzyme immunoassay (De Saeger et al. 2002), fluorescence polarization
immunoassay (Maragos and Plattner 2002), immunosensors (Alarcon
 Ochratoxin A in Some French Wines 1189

et al. 2006), and biosensors (van der Gaag et al. 2003). Immunoassay
methods exhibit good sensitivity and acceptable performance, allowing
the detection of OTA at concentrations equal or less than the EC
regulatory values. Several research papers based on traditional spectro-
photometric enzyme–linked immunosorbent assays (ELISA) have been
already published (Ramakrishna et al. 1990; Clarke et al. 1993; Barna
Vetrò et al. 1996; Kwak and Shon 2000; Alarcon et al. 2006; Liu et al.
2008; Wang et al. 2007; Cho et al. 2005; Fujii et al. 2006). Enzyme-linked
immunosorbent assay is well suited for specific and relatively inexpensive
screening of OTA in wine, and the increasing demand from the industry
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for screening tests for in-field use is challenging. Although immunochem-

ical methods have become very popular in recent years, they require a
stable source of antibodies, mainly achieved from commercial sources.
Affordable antigen–enzymatic conjugates continue to be less commonly
sold independent of commercial ELISA-based test kits, and for this rea-
son easy conjugation and rapid purification procedures are required for
the future development of immunochemical assays. Moreover, chemico-
physical and analytical investigations and characterization are necessary
to ascertain the chemicophysical properties of such bioconjugates.
In this research article, we focused on the chemicophysical and
analytical characterization of an OTA–horseradish peroxidase (HRP)
conjugate (OTA–HRP) obtained by linking covalently the 1,10 -carbonyl-
diimidazole activated OTA to HRP. Ultraviolet-vis, fluorescence, sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE),
Western blot, and ELISA tests were performed to investigate the
OTA-HRP conjugate. Commercially and noncommercially available
wine samples were assayed by a direct competitive ELISA approach,
and the obtained results were confirmed by HPLC analysis.

MATERIALS, REAGENTS, METHODS, AND APPARATUS

Materials and Reagents

Ochratoxin A (O 1877), 3,30 ,5,50 -tetramethylbenzidine (TMB, T0440)

liquid substrate for ELISA, glycerol (G 7893), bovine seric albumin
(BSA, A9647), HRP (P 8375, 3.2 RZ profile), acrylamide=bis-acrylamide
(A 7802), magnesium chloride (M 8266), glycine (G 8898) dodecyl sulfate
sodium salt (L 3771), casein (C 7594), potassium phosphate monobasic (P
0662), potassium phosphate dibasic (P3786), potassium chloride (P 3911),
Trizma base (T-1503), 5-bromo-4-chloro-3-indolyl phosphate dipotas-
sium salt (B 6274), and Coomassie brilliant blue G (B 0770) were
purchased from Sigma. Sodium carbonate (223530) was purchased
 1190 A. Radoi et al.

from Aldrich. Sodium bicarbonate (S6014), sodium chloride (S 7653),

Tween 20 (274348), p-nitrotetrazolium blue (N 6876), and nitrocellulose
membranes for immunoblotting (N 789) were purchased from Sigma-
Aldrich. Acetone (P0050222) and methanol (414816) were supplied from
Carlo Erba Reactifs-SDS. 1,10 -Carbonyldiimidazole (21860) was from
Fluka. Illustra NAP-10 columns prepacked with Sephadex G-25 DNA
grade in distilled water containing 0.15% Kathon CG=ICP biocide
(17-0854-01) were from GE Healthcare. NuPage LDS buffer (NP 0007)
was from Invitrogen. Polystyrene microtiter plates (MaxiSorp) were pur-
chased from NUNC (Roskilde, Denmark). A Multiskan EX (Thermo
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Life Sciences, Cergy-Pontoise, France) microplate photometer was

utilized for ELISA colorimetric measurements. All other reagents not
specified were from Sigma or Sigma-Aldrich.

OTA–HRP Conjugate: Conjugation Reaction, Purification, and Storage

The conjugation protocol was based on a modified version described by

Xiao et al. (1995). The optimized conjugation protocol, used to covalently
link the HRP to OTA was the following: 5 mg of OTA dissolved in 500 mL
of acetone were added to 7.5 mg of 1,10 -carbonyldiimidazole (CDI) and
stirred in an amber vial for 20 min at ambient temperature. This reaction
mixture was added drop by drop to an enzymatic solution of HRP (21 mg
dissolved in 2 mL of carbonate buffer, pH 9.8), and this reaction was
allowed to proceed for 4 h at ambient temperature under stirring and pro-
tected from light. The reacted OTA was separated from the unconjugated
OTA by passing aliquots (500 mL) of this final reaction mixture through
Illustra NAP-10 columns, with a mobile phase of phosphate buffer saline
(PBS, 15 mM, pH 7.4). Only the brownish fractions were collected for a
total volume of 5.0 mL. Collected fractions were stored at 4 C.

UV-Vis and Fluorescence Investigation

For UV-vis and fluorescence investigations, undiluted and properly

diluted (in PBS, 15 mM, pH 7.4) aliquots of OTA–HRP conjugate were
used. Unconjugated HRP and OTA were used as references, diluted also
in PBS. UV-vis analysis was performed in the 240- to 600-nm range.
Fluorescence emissions spectra were obtained either at 333 nm (charac-
teristic for OTA) or at 295 nm (specific for tryptophan). UV-vis spectro-
photometric measurements were performed using a Hewlett Packard
diode array 8451A spectrophotometer. SAFAS flx-Xenius fluorimeter
was used during fluorescence measurements.
 Ochratoxin A in Some French Wines 1191

Gel Electrophoresis and Western Blot Analysis

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE),

using 5% stacking gel and 12% separating gel, was performed according to
the methods described by Laemmli (1970). A vertical gel electrophoresis
unit (Mini-Protean II; Bio-Rad Laboratories, Richmond, CA) was used,
and the gels were run at 200 V constant voltage, during 1 h, in tris-
glycine-SDS (10X) running buffer. Different dilutions (1=2, 1=4, 1=10 v=v
and undiluted as collected from the Illustra NAP-10 column) of OTA–
HRP conjugate were mixed with 4X NuPage LDS buffer and loaded on
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the SDS–PAGE gel, with the native HRP (15 mg) as control. Gels were then
either stained with Coomassie blue (0.125% w=v Coomassie blue in 40% v=v
methanol and 10% v=v acetic acid for 1 h, then destained in 40% v=v metha-
nol and 10% v=v acetic acid) or further processed for Western blotting.
After SDS–PAGE, proteins were transferred to the nitrocellulose
membrane using a semidry transfer method (Bio-Rad, semidry blotter,
5.5 mA=cm2) for 45 min at 10 V. Following transfer, the membrane was
blocked (30 min) by incubation with 1.5% w=v caseine at room temperature
in PBS and then incubated with diluted anti-OTA antibody (2.5 mL in PBS
þ0.05% v=v Tween 20, 5 mg=mL) for 1 h at room temperature with constant
shaking. After incubation with anti-OTA antibody, the membrane was incu-
bated with 1=5000 v=v antimouse phosphatase-labeled antibody prepared
in PBS for 60 min at room temperature. In between each step, washings
were performed using PBS þ0.05% v=v Tween 20. Before detection, the
membrane was washed extensively and developed by conventional methods
with nitroblue tetrazolium (NBT)=5-bromo-4-chloro-3-indolylphosphate
(BCIP) in 100 mM Tris, 100 mM NaCl, and 5 mM MgCl2 (pH 9.5).

Wine Samples, Sample Preparation, and Immunoaffinity Cleanup

Several wines were purchased from local stores or were kindly provided
by small local wine producers. A total of seven samples were analyzed:
three red and four white wines.
For ELISA and HPLC analysis, 10.0 mL of wine (red or white) were
mixed with 3 mL of 50% v=v methanol solution prepared in distilled water,
and the pH was adjusted to 7.4 with Trizma base buffer solution (2 M, ca.
5.5 mL to each 10 mL of wine), and all was brought to a final volume of
20 mL with distilled water. The 20 mL of sample were passed through an
immunoaffinity column (OCHRAPREP, R-Biopharm Rhône Ltd.) at a
constant speed of 0.8 mL=min using a peristaltic pump (Miniplus 3, Gilson,
France). The column was washed with 20 mL of PBS (15 mM, pH 7.4) at
1.2 mL=min. The immobilized OTA was eluted with 1-mL fractions of
 1192 A. Radoi et al.

methanol (flow rate 1 mL=min), back-extracting twice for each 1-mL metha-
nol fraction, for a total volume of 3.0 mL. The eluted OTA was evaporated
to dryness under a nitrogen stream and reconstituted up to 3.0 mL with the
mobile phase (for HPLC analysis) or up to 5.0 mL with PBS (for ELISA).

Spectrophotometric ELISA

In the direct competitive ELISA (dc-ELISA), the 96-well ELISA plate

(MaxiSorp) was coated with anti-OTA antibodies (2 and 1 mg=mL). The
coating step (100 mL=well, 4 C, ON) was performed in 50 mM carbonate
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buffer (CB), pH 9.8. The blocking step (30 min) was performed at 22 C
(rt), using 150 mL=well of 1% w=v BSA solution (prepared in 15 mM PBS,
pH 7.4). The buffer competition was allowed to proceed for 60 min at rt by
adding nonlabeled OTA (50 mL) and OTA–HRP conjugate (50 mL,
1=2500 v=v), all prepared in PBS, to the inside of the wells. For real sample
analysis, 50 mL of the 5.0-mL reconstituted immunoaffinity columns cleaned
up with wine or properly diluted in PBS to fit inside the linear working range
were mixed with equal volumes of OTA–HRP conjugate (1=2500 v=v in PBS)
during the competition step, up to a total volume of 100 mL. Finally, 100 mL of
3,30 ,5,50 -tetramethylbenzidine liquid substrate for ELISA were added in each
well, and the absorbance (620 nm) was read after 10 min. Washing
(3  200 mL) was performed after each step using a solution of 0.05% v=v of
Tween 20 prepared in PBS (2  200 mL) followed by only PBS (1  200 mL).
The binding curve was obtained in a similar way as described before for
the dc-ELISA, but in the coating step only 2 mg=mL of anti-OTA antibody
were used and only serial dilutions in PBS of the OTA–HRP were utilized.
Spectrophotometric ELISA standard curves were performed in
triplicate, and the mean of each value was used for curve fitting. The
calibration curves (absorbance at 620 nm vs. antigen concentration or con-
jugate serial dilutions) were fitted using nonlinear four-parameter logistic
calibration plots (Warwick 1996). The data obtained for each curve were
plotted and fitted using SigmaPlot software (SPSS), and a regression
analysis on the linear portion of the sigmoidal curves was also performed.
For binding and competition steps, the 5G9 clone from Soft Flow
Biotechnology Ltd., Gödöll} o, Hungary, was utilized. An anti–aflatoxin
M1 antibody (3G11, Soft Flow Biotechnology Ltd., Gödöll} o, Hungary)
was utilized as reference in the binding assays.

HPLC Analysis

Detection of OTA was achieved by using 333-nm excitation and 443-nm

emission wavelengths. A 100-mL portion of the reconstituted extract was
 Ochratoxin A in Some French Wines 1193

injected into the HPLC apparatus (L-6220 Intelligent Pump, Merk,

Germany) equipped with a fluorescence detector (RF-10Al, Shimadzu,
Kyoto, Japan) and a Merck D-2500 Chromato-Integrator recorder. A
5-mm Spherisorb ODS-2 (150  4.6 mm) HPLC column was utilized,
and an acetonitrile–water–acetic acid (51:47:2 v=v=v) mobile phase was
pumped constantly at a flow rate of 1.0 mL=min. The OTA was identified
by constant retention time and quantified by comparison with peak areas
of known standards in the mobile phase. Acetonitrile (P00637G21,
HPLC gradient), methanol (414816), and acetic acid glacial (401422)
were all from Carlo Erba Reactifs-SDS.
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RESULTS AND DISCUSSION

UV-Vis and Fluorescence Investigation of OTA–HRP Conjugate

Horseradish peroxidase is a monomeric glycoprotein, with a single

polypeptide chain consisting of 308 residues; the complete amino acid
sequence was determined by Welinder (1979). From the 308 residues,
26 are aromatic [5 tyrosine (Tyr), 1 tryptophan (Trp), and 20 phenyla

Figure 1. UV-vis absorption spectrum of HRP (0.58 mg=mL, solid line) and
OTA–HRP (collected fraction from the Illustra NAP-10 column was diluted
1=4 v=v, dashed line) in PBS, 15 mM, pH 7.4.
 1194 A. Radoi et al.

lanine (Phe)]. When we compared the native HRP UV-vis absorption

spectrum with the OTA–HRP conjugate spectrum (Fig. 1, OTA–HRP
conjugate diluted 1=4 v=v in PBS), we observed that the absorption bands
(270 and 280 nm) due to the aromatic amino acid residues in OTA–HRP
conjugate increased, as well as the 404 nm band (attributed to the
prosthetic heme). This suggested that there was a change in the micro-
environment surrounding the Trp and Tyr residues and also that the
hemin content in the conjugate changed from a calculated 3.0 to 2.2 RZ
profile; the loss was attributed to the organic solvent (acetone) utilized
during the conjugation procedure. The concentration of HRP in the
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1=4 v=v diluted OTA–HRP conjugate was calculated to be 0.61 mg=mL

(e403 ¼ 102 000 M
1cm
1) (Ohlsson and Paul 1973); meanwhile the HRP
concentration used in the UV–vis experiments was 0.58 mg=mL.
In the HPR molecule, both tryptophan and tyrosine residues show
fluorescence, but the fluorescence emission excited at 295 nm solely arises
from the tryptophan residue (Ugarova, Savitski, and Berezin 1981).
From UV-vis investigations, we observed that bands usually attributed
to aromatic amino acid residues present in proteins were altered when
confronted with the native HRP enzyme. Moreover, the change in the

Figure 2. Normalized fluorescence spectra of native HRP (1.0 mg=mL, solid line)
and OTA-conjugated HRP (undiluted, as collected from the Illustra NAP-10
column, dashed line) in PBS, 15 mM, pH 7.4; excitation wavelength 295 nm.
 Ochratoxin A in Some French Wines 1195
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Figure 3. Normalized fluorescence spectra of OTA (dashed line) and OTA-

conjugated HRP (solid line) in PBS, 15 mM, pH 7.4; excitation wavelength 333 nm.

microenvironment surrounding the Trp residue can change the emission

maximum of the tryptophan fluorescence (Chattopadhyay and Mazumdar
2000). Figure 2 indicates that the Trp fluorescence emission shifted from
326 (in the native HRP) to 318 nm after HRP was conjugated to OTA, with
the blue shift reflecting the reduced accessibility of tryptophan residue to
the bulk solvent (Mogharrab, Ghourchain, and Amininasab 2007).
Ochratoxin A is a fluorescent molecule (Frenette et al. 2008), and
when excited at 333 nm, it shows a characteristic emission peak at
438 nm. When the OTA–HRP conjugate was excited at the same excita-
tion wavelength used for the unconjugated OTA, we observed a peak shift
from 438 to 444 nm (Fig. 3), this red shift being attributed to a structural
change in the excited state of conjugated OTA (Frenette et al. 2008).
The change in fluorescence of the linked OTA and the fluorescence
blue shift of the tryptophan residue, along with the UV-vis investigation,
confirmed that the OTA is covalently linked to the HRP.

SDS–PAGE and Western Blot Results

Coomassie blue–stained gel showed similar patterns for OTA–HRP

(diluted and undiluted conjugate) and native HRP (Fig. 4A), because
 1196 A. Radoi et al.
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Figure 4. HRP–OTA conjugate undiluted (ND) and diluted 1=2, 1=4, and
1=10 v=v and HRP (15 mg) were separated on 12% SDS–PAGE and stained with
Coomasie blue (A) or transferred to nitrocellulose membrane and immunoblotted
using anti-OTA antibodies and antimouse phosphatase-labeled antibodies (B).

the OTA molecule (MW ¼ 403.81) does not produce a mass increase suf-
ficient enough to discriminate from native HRP. Surprisingly, the band
size corresponding to OTA–HRP conjugate was relatively increased with
respect to the native HRP reference band. Plausible explanations can be
attributed either to the fact that the SDS–PAGE assay was performed in
nondenaturing conditions (the samples were not heated before running
SDS–PAGE) or to the fact that the native HRP is constituted from at
least seven isozymes (http://www.sigmaaldrich.com/sigma/datasheet/
p8375dat.pdf). More probable is that during conjugation, dimmers, tri-
mers, tetramers, or other ‘‘multimers’’ of OTA–HRP were formed. All
these possible explanations helped us to understand the Western
blot–developed membrane (Fig. 4B), because there were four different
visible bands corresponding to the undiluted, 1=2, 1=4, and 1=10 v=v
diluted OTA–HRP conjugate. As expected, no band(s) corresponded to
native HPR. The absence of band(s) in the native HRP line indicated
without a doubt that the anti-OTA antibodies were bound only to the
corresponding lines where OTA–HRP conjugate was loaded. Finally,
the presence of different bands in each running line was more probably
attributable to different ‘‘multimers’’ of OTA–HRP formed during
the conjugation of OTA with HRP, because the coupling reagent (1,10 -
carbonyldiimidazole), when used in excess, can generate secondary
reactive products.
 Ochratoxin A in Some French Wines 1197

Spectrophotometric ELISA and Wine Analysis

Generally, ELISA procedures require the investigation of parameters

such as primary and=or secondary antibody concentration=dilution,
conjugate dilution=concentration, temperature, incubation time, and so
on. For our study, it was important to assess the optimum dilution of
the OTA–HRP used during the competition step and also to investigate
if the OTA–HRP conjugate specifically binds only the anti-OTA antibo-
dies. As illustrated in Fig. 5, it is evident that the conjugate specifically
binds the anti-OTA antibody (5G9 clone); meanwhile, a nonspecific
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adsorption=binding is only present at concentrated values of conjugate.

We used anti–aflatoxin M1 antibodies as reference antibodies to investi-
gate the specific interaction. As can be seen in Fig. 5, a ‘‘hook effect’’
manifested during binding assays as a result of the low dilution ratio of
the OTA–HRP conjugate, which saturated the available binding sites.
Optimization of the direct competitive immunoassay parameters
required the study of the effect of the antibody loading during the coating
step, because this is the key step when performing a dc-ELISA. Three dif-
ferent antibody concentrations were investigated (2, 1, and 0.5 mg=mL),
but we obtained relevant information for only two of them (Fig. 6).

Figure 5. Binding curve obtained from serial dilutions (in PBS, 15 mM, pH 7.4) of
the OTA–HRP conjugate; 2 mg=mL anti–ochratoxin A antibody and 2 mg=mL
anti–aflatoxin M1 (used for the blank tests).
 1198 A. Radoi et al.
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Figure 6. Competition curve performed in PBS, 15 mM, pH 7.4; 1% w=v BSA in

the blocking step, OTA–HRP (1=5000 v=v inside the wells).

For the 0.5 mg=mL antibody coating concentration, we had random

results, attributed to insufficient coverage of the wells. In the competition
step, the OTA–HRP was diluted 1=2500 v=v. When 2 mg=mL of 5G9 anti-
body were utilized, the IC50 was 1200 ng=L, with a linear working range
(LWR) between 400 and 3000 ng=L. For 1 mg=mL of anti-OTA antibody

Table 1. Comparison of HPLC and dc-ELISA data analysis

Concentration (mg=L)

Wine Color HPLC ELISA RE (%)

Maury Blanc 1 White 0.31 0.36 þ16

Maury Rouge 1 Red 0.44 0.52 þ18
Maury Rouge 2 Red 0.74 0.85 þ15
Cotes du Roussillon Red 0.91 1.1 þ21
Wine 1 White 0.59 0.67 þ14
Wine 2 White 0.56 0.70 þ25
Wine 3 White 0.92 1.1 þ19

Note. The first four types of wine were commercially available;

the remaining three were obtained from local wine producers.
 Ochratoxin A in Some French Wines 1199

utilized in the coating step, the IC50 value was three times less (400 ng=L),
and the LWR ranged from 100 to 1500 ng=L. These parameters (competi-
tive value of IC50 and a wide LWR) fulfill the requirements necessary to
detect OTA in real samples. Direct-competitive ELISA (dc-ELISA) wine
analysis performed using the OTA–HRP conjugate illustrated good agree-
ment with HPLC analysis as shown in Table 1. Also, the 5G9 antibody has
an ochratoxin B (OTB) cross reactivity of 9%, which could explain why
dc-ELISA constantly overestimated the amount of OTA in the wine samples.

CONCLUSIONS
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In this study, we characterized OTA–HRP, obtained by covalently

linking the 1,10 -carbonyldiimidazole-activated OTA to HRP. The analy-
tical characteristics of this conjugate were investigated by means of fluor-
escence, UV-vis spectrophotometry, SDS–PAGE, Western blot, and
immunoassay. Instrumental analysis confirmed that the bound OTA
red-shifted in the emission in 6 nm fluorescence and also that the micro-
environment surrounding the Trp residue was altered. Electrophoresis
experiments confirmed that the migration band of the OTA–HRP conju-
gate is at the same level as for the native HRP; meanwhile, the size of the
bands were not so sharp, suggesting the presence of secondary reaction
products, which were confirmed by immunoblotting. Western blot assay
confirmed that anti-OTA antibodies specifically bound the OTA–HRP
conjugate, because no band was present in the native HRP reference run-
ning line. A dc-ELISA was developed using the OTA–HRP conjugate,
and several types of red and white French wines were analyzed. The
obtained results were confirmed by HPLC analysis.

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