Hapten synthesis and monoclonal antibody-based immunoassay development for the detection of the fungicide kresoxim-methyl

Article abstract of DOI:10.1021/jf073039x

Strobilurin fungicides have been increasingly used for fungus pest control since they were introduced in 1996. For pesticide residue detection, immunoassays constitute nowadays a valuable approach. This paper describes the synthesis of functionalized haptens of kresoxim-methyl, the production of monoclonal antibodies, and the development of enzyme-linked immunosorbent assays. On the one hand, a two-step conjugate-coated immunoassay was optimized using extended or short incubation times, with limits of detection of 0.4 ng/mL for the extended assay and 0.3 ng/mL for the rapid assay. On the other hand, an immunoassay was optimized following a procedure consisting of just one incubation step. This one-step assay had a limit of detection of 0.4 ng/mL. All of these assays showed a similar performance, with sensitivities well below common maximum residue limits for this pesticide (50 μg/kg) and lower than the detection limits of the usual chromatographic detection methods.

Full text of DOI:10.1021/jf073039x

J. Agric. Food Chem. 2008, 56, 1545–1552 1545
Hapten Synthesis and Monoclonal Antibody-Based
Immunoassay Development for the Detection of the
Fungicide Kresoxim-methyl
JOSEP V. MERCADER,† CELIA SUÁREZ-PANTALEÓN,† CONSUELO AGULLÓ,§
ANTONIO ABAD-SOMOVILLA,§ AND ANTONIO ABAD-FUENTES*,†
Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo
Superior de Investigaciones Científicas (CSIC), P.O. Box 73, 46100 Burjassot, València, Spain, and
Departamento de Química Orgánica, Universitat de València, C/ Doctor Moliner 50,
46100 Burjassot, València, Spain
Strobilurin fungicides have been increasingly used for fungus pest control since they were introduced
in 1996. For pesticide residue detection, immunoassays constitute nowadays a valuable approach.
This paper describes the synthesis of functionalized haptens of kresoxim-methyl, the production of
monoclonal antibodies, and the development of enzyme-linked immunosorbent assays. On the one
hand, a two-step conjugate-coated immunoassay was optimized using extended or short incubation
times, with limits of detection of 0.4 ng/mL for the extended assay and 0.3 ng/mL for the rapid assay.
On the other hand, an immunoassay was optimized following a procedure consisting of just one
incubation step. This one-step assay had a limit of detection of 0.4 ng/mL. All of these assays showed
a similar performance, with sensitivities well below common maximum residue limits for this pesticide
(50 µg/kg) and lower than the detection limits of the usual chromatographic detection methods.
KEYWORDS: Strobilurin; ELISA; dipeptide; linker; spacer arm; bridge; hapten heterology; pesticide; QoI
fungicides
INTRODUCTION
developed countries because they all are relatively readily
degraded and represent low risk to human health as well as to
birds, mammals, and bees, although they vary in their toxicity
to aquatic organisms (7). Nevertheless, fungus resistance to
strobilurins has already been reported (8), so the Fungicide
Resistance Action Committee (FRAC) has recommended pre-
ventative application for optimum disease control (www.
frac.info). Moreover, their extensive use and postharvest
application makes it easier to reach human consumers. These
facts have led the regulatory organisms to introduce controls
for strobilurin residues in food products (9).
The discovery of the strobilurin pesticides was inspired by a
group of natural antibiotics produced by a range of Basidi-
omycete wood-rooting fungi. In 1977, Anke and co-workers
(1) obtained two compounds from fermentations of Strobilurus
tenacellus, strobilurins A and B (Figure 1), with a powerful
antibiotic activity against a range of fungal species. It was
observed that these molecules contained a common element
essential to their mode of action, a ꢀ-methoxyacrylate subunit
(2). Since those years, several synthetic derivatives of the
strobilurin family have been prepared and commercialized, all
of them incorporating the same, or slightly modified, toxophore
moiety. Kresoxim-methyl (KM), with a ꢀ-methoxyiminoacetate
group (Figure 2), was one of the first strobilurins to be patented
(3) and, in 1999, it was included in Annex I of the EU Council
Directive 91/414/EEC as an approved nonsystemic active
substance for plant protection (4). Nowadays, strobilurins,
classified as QoI fungicides, are extensively used for crop and
culture protection and constitute a major class of agricultural
fungicides; they represent approximately 10% of the fungicide
market (5, 6). These chemicals have been approved for use in
Current analytical methods for the detection of KM consist of
extensive organic solvent extraction followed by gas chromatog-
raphy analysis with nitrogen-phosphorus detection (10), although
other chromatographic methods have been reported (11, 12).
Biotechnology-based methods such as immunoassays (IAs) are
an alternative or complementary tool for pesticide monitoring
(13). Since they were introduced, many IAs have been described
for pesticides of different families. Most IAs have been
developed using polyclonal antisera raised in rabbits because
the production of monoclonal antibodies (mAbs) has usually
been considered to be an expensive and laborious technology.
However, as IAs have been transferred to the industry, this initial
cost becomes less important, considering the subsequent expense
of assay validation and the potential of an unlimited supply of
uniform immunoreagents.
* Author to whom correspondence should be addressed [telephone
(+34)-96-3900022; fax (+34)-96-3636301; e-mail aabad@iata.csic.es].
† Consejo Superior de Investigaciones Científicas.
§ Universitat de València.
10.1021/jf073039x CCC: $40.75
2008 American Chemical Society
Published on Web 02/06/2008

1546 J. Agric. Food Chem., Vol. 56, No. 5, 2008
Mercader et al.
Figure 2. Scheme of hapten synthesis and structures of kresoxim-methyl
(KM) and functionalized derivatives.
Figure 1. Structures of the natural strobilurin fungicides produced by
Basidiomycetes fungi: (A) strobilurin A; (B) strobilurin B. The common
(E)-methyl ꢀ-methoxyacrylate group acting as toxophore is circled.
(TLC) using 0.25 mm precoated silica gel plates. Visualization was
carried out with UV light and aqueous ceric ammonium molybdate
solution or 50% (v/v) concentrated H2SO4 in water. Chromatography
refers to flash column chromatography and was carried out with the
indicated solvents on silica gel 60 (particle size ) 0.040–0.063 mm).
All melting points were determined using a Kofler hot-stage apparatus
and are uncorrected. All NMR spectra were recorded in CDCl3 or
DMSO-d6 at room temperature on a Bruker AC-300 spectrometer
(300.13 MHz for 1H and 75.47 MHz for 13C). The spectra were
referenced to residual solvent protons in the 1H NMR spectra (7.26
and 2.50 ppm) and to solvent carbons in the 13C NMR spectra (77.0
and 39.43 ppm). Carbon substitution degrees were established by DEPT
pulse sequences. A combination of COSY and HSQC experiments was
In 2006, a research paper was published about the develop-
ment of three highly sensitive IA formats using selective
polyclonal antibodies for the detection of azoxystrobin (14),
another member of this fungicide family. To our knowledge,
no additional papers have appeared describing the development
of IAs to any other member of the strobilurin family, and no
mAbs have been obtained so far to any strobilurin. In this paper,
we report a strategy for the synthesis of functionalized haptens
for the methoxyiminoacetate strobilurins and the production of
high-affinity monoclonal antibodies against KM. Three enzyme-
linked immunosorbent assays (ELISAs) with different proce-
dures and operation times for KM detection have been optimized
and characterized.
1
utilized for the assignment of H and 13C chemical shifts. IR spectra
were measured as thin films for liquid compounds and as KBr pellets
for solids using a Nicolet Avatar 320 spectrometer. High-resolution
mass spectra were recorded with a VG AutoSpec spectrometer.
Hapten Synthesis. All KM derivatized haptens that were prepared
in this study are summarized in Figure 2. Haptens were synthesized
by alkaline hydrolysis of the KM ester, activation of the resulting
carboxylic acid with DCC and NHS, and reaction with an amino acid
(4-aminobutanoic acid and 6-aminohexanoic acid) or a dipeptide (N-
glycyl-glycine, N-ꢀ-alanyl-glycine, and N-glycyl-γ-aminobutyric acid).
Compounds used in this study present minor safety concerns. However,
it is advisable to work in a well-ventilated fume hood during synthesis
work.
MATERIALS AND METHODS
Chemicals and Instrumentation. Kresoxim-methyl [methyl (E)-
2-methoxyimino-2-[2-(o-tolyloxymethyl)phenyl]acetate] (CAS Registry
No. 143390-89-0), dimoxystrobin, and pyraclostrobin, Pestanal grade,
were purchased from Riedel-de-Haën (Seelze, Germany). Technical
grade KM was generously provided by BASF (Limburgerhof, Ger-
many). Trifloxystrobin standard was kindly provided by Bayer Crop-
Science (Frankfurt, Germany), and azoxystrobin and picoxystrobin were
provided by Syngenta (Basel, Switzerland). Stock solutions of these
compounds were prepared in dried N,N-dimethylformamide (DMF) and
stored at -20 °C. N-Glycyl-glycine (gg) was obtained from Alfa Aesar
(Karlsruhe, Germany), and N-ꢀ-alanyl-glycine (ꢀag) and N-glycyl-γ-
aminobutyric acid (gab) were from Bachem AG (Bubendorf, Switzer-
land). N,N-Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide
(NHS), 4-aminobutyric acid, 6-aminohexanoic acid, and tributylamine
were from Fluka (Madrid, Spain). Isobutyl chloroformate, ovalbumin
(OVA), thimerosal, and o-phenylenediamine (OPD) were purchased
from Sigma-Aldrich (Madrid, Spain). Sephadex G-25 HiTrap Desalting
columns and HiTrap Protein G HP columns from General Electric
Healthcare (Uppsala, Sweden) were used for conjugate and antibody
purification, respectively. Polyclonal rabbit antimouse immunoglobulins
peroxidase conjugate (RAM-HRP) was from Dako (Glostrup, Den-
mark). Bovine serum albumin (BSA) fraction V and Hybridoma Fusion
and Cloning Supplement (HFCS) were purchased from Roche Applied
Science (Mannheim, Germany). P3-X63-Ag 8.653 mouse plasmacytoma
cell line was from the European Collection of Cell Cultures (Wiltshire,
U.K.). HT and HAT supplements and gentamicine solution were
obtained from Gibco BRL (Paisley, Scotland). Cell culture media (high-
glucose Dulbecco’s Modified Eagle’s Medium), poly(ethylene glycol)
(PEG1500), fetal bovine serum (FBS), 200 mM alanyl-glutamine
solution, Red Blood Cell Lysing Buffer Hybri-Max, MEM nonessential
amino acid solution, and Freund’s adjuvants were from Sigma-Aldrich
(Madrid, Spain). Culture plasticware and Costar flat-bottom high-
binding polystyrene ELISA plates were from Corning (Corning, NY).
Ultraviolet–visible spectra and ELISA absorbances were read (in dual
wavelength mode, 492–650 nm) with a PowerWave HT from BioTek
Instruments (Winooski, VE). ELISA plates were washed with an
ELx405 microplate washer also from BioTek Instruments.
(E)-2-(Methoxyimino)-2-(2-(o-tolyloxymethyl)phenyl)acetic Acid
(KM0). Five grams of KM (15.96 mmol), methanol (70 mL), and 5 M
aqueous sodium hydroxide (10.0 mL) were stirred at reflux. After the
hydrolysis was complete, as monitored by TLC (around 5 h), most of
the methanol was evaporated off under reduced pressure, water was
added to the residue, and neutral impurities were removed by extraction
with ethyl acetate. The aqueous layer was cooled to 0 °C and acidified
to pH 3 with 2 M HCl aqueous solution, the product was extracted
with ethyl acetate, and the extract was washed with brine and finally
dried over anhydrous MgSO4. Evaporation gave 4.09 g of a white solid
(85.6%), which was used in the next steps without further purification.
The preparation of this acid derivative has been described in the
literature (15, 16), but no physical or spectroscopic data have been
previously given for it. A detailed description of these data follows:
mp 139–140 °C (from slow evaporation of a CHCl3/hexane solution);
1H NMR (DMSO-d6), δ 7.43 (1H, dd, J ) 7.5 and 1.5 Hz, H-3 Ph),
7.30 (1H, ddd, J ) 7.5, 7.5, 1.5 Hz, H-4 Ph), 7.25 (1H, ddd, J ) 7.5,
7.5, 1.5 Hz, H-5 Ph), 7.15–7.06 (3H, m, H-6 Ph, H-3 tolyl and H-5
tolyl), 6.87 (1H, br d, J ) 7.5 Hz, H-6 tolyl), 6.81 (1H, dd, J ) 7.5
and 7.5 Hz, H-4 tolyl), 4.98 (2H, s, CH2O), 3.76 (3H, s, OCH3), 2.22
(3H, s, CH3-tolyl); 13C NMR (DMSO-d6), δ 164.70 (CO), 156.31 (C-
2), 156.10 (C-1 tolyl), 134.44 (C-2 Ph), 133.01 (C-1 Ph), 130.32 (C-3
tolyl), 127.53 (C-6 Ph), 127.22 (C-5 tolyl), 126.81 (C-3 Ph), 126.56
(C-4 Ph), 125.79 (C-2 tolyl), 125.72 (C-5 Ph), 120.14 (C-4 tolyl), 111.34
(C-6 tolyl), 66.96 (CH2O), 61.31 (CH3O), 16.11 (CH3); IR (KBr), 3472,
3070, 3025, 2977, 2943, 1710, 1616, 1494, 1377, 1251, 1008, 757,
744 cm-1; MS (EI), m/z (%) 299 (M+, 4), 268 (1), 223 (32), 192 (15),
116 (100), 89 (11); HRMS, calcd for C17H17NO4 299.11576, found
299.11652; UV (100 mM sodium phosphate buffer, pH 7.4), ꢀ (280
Reagents for hapten synthesis were obtained from commercial
sources and used without purification. Tetrahydrofuran was distilled
from sodium and benzophenone under an argon atmosphere. The
reactions were monitored with the aid of thin-layer chromatography
nm) ) 1.37 mM-1 cm-1, ꢀ (260 nm) ) 2.86 mM-1 cm-1
.
General Procedure for Spacer Arm Introduction. The carboxylic acid
(KM0) (1.0 mmol), NHS (1.1 mmol), and DCC (1.1 mmol) were
dissolved in dry tetrahydrofuran (5.5 mL) at 0 °C under nitrogen

Immunoassays to Kresoxim-methyl
J. Agric. Food Chem., Vol. 56, No. 5, 2008 1547
atmosphere. The mixture was stirred at 4 °C for 20 h and then at room
temperature for 4 h. The precipitated N,N′-dicyclohexylurea was
removed by vacuum filtration and a solution of 1.1 mmol of NaHCO3,
and 1.1 mmol of the corresponding amino acid or dipeptide in 1.8 mL
of water was added. The reaction mixture was stirred at room
temperature until TLC (CHCl3/CH3OH 5:1) showed complete con-
sumption of the intermediate NHS ester (around 40 h); then the solvent
was evaporated off, and the obtained residue was dissolved in 10%
(w/v) aqueous citric acid and extracted with ethyl acetate. The combined
organic layers were washed with brine, dried over anhydrous Na2SO4,
and evaporated under reduced pressure to afford the crude coupling
product, which was purified by column chromatography on silica gel
using the eluent specified in each case.
7.37 (1H, ddd, J ) 7.5, 7.5, 1.5 Hz, H-4 Ph), 7.20 (1H, dd, J ) 7.5,
1.5 Hz, H-3 Ph), 7.13 (1H, br d J ) 7.5 Hz, H-3 tolyl), 7.09 (1H, br
t, J ) 8 Hz, H-5 tolyl), 6.89 (1H, br d, J ) 8 Hz, H-6 tolyl), 6.82 (1H,
dd, J ) 7.5 and 7.5 Hz, H-4 tolyl), 4.92 (2H, s, CH2O), 3.92 (3H, s,
OCH3), 3.83 (2H, d J ) 5.8 Hz, H-7), 3.77 (2H, d J ) 5.8 Hz, H-10),
2.16 (3H, s, CH3-tolyl); 13C NMR (DMSO-d6), δ 171.07 (C-11), 168.62
(C-8), 162.17 (C-5), 156.02 (C-1 tolyl), 150.67 (C-4), 135.41 (C-2 Ph),
130.36 (C-3 tolyl), 129.36 (C-1 Ph), 129.01 (C-5 Ph), 128.65 (C-3 Ph),
127.15 (C-6 Ph), 127.05 (C-4 Ph), 126.79 (C-5 tolyl), 125.81 (C-2 tolyl),
120.29 (C-4 tolyl), 111.26 (C-6 tolyl), 67.18 (CH2O), 62.77 (CH3O),
41.99 (C-7), 40.54 (C-10), 15.86 (CH3); IR (KBr), 3332, 3067, 2938,
1663, 1601, 1522, 1495, 1443, 1241, 1123, 1054, 1021, 983, 752 cm-1
;
MS (EI), m/z (%) 414 (M+ + 1, 0.5), 413 (M+, 0.5), 383 (5), 382
(21), 307 (19), 306 97), 275 (11), 223 (10), 203 (11), 173 (8), 117
(17), 116 (100); HRMS, calcd for C21H23N3O6 413.15869, found
413.15922; UV (100 mM sodium phosphate buffer, pH 7.4), ꢀ (280
(E)-6-2-(Methoxyimino)-2-(2-(o-tolyloxymethyl)phenyl)acet-
amido)hexanoic Acid (KM6). Purification of the crude coupling
product obtained from 2.52 g of KM0 by column chromatography,
eluting with CHCl3/EtOAc 8:2, afforded 1.75 g of pure KM6 (50%
yield) as a viscous oil: 1H NMR (CDCl3), δ 7.54 (1H, dd, J ) 7.4, 1.4
Hz, H-6 Ph), 7.42 (1H, ddd, J ) 7.4, 7.4, 1.6 Hz, H-5 Ph), 7.37 (1H,
ddd, J ) 7.4, 7.4, 1.5 Hz, H-4 Ph), 7.23 (1H, dd, J ) 7.4, 1.5 Hz, H-3
Ph), 7.15–7.08 (2H, m, overlapped H-3 tolyl and H-5 tolyl), 6.85 (1H,
dd, J ) 7.0 and 7.0 Hz, H-4 tolyl), 6.79 (1H, br d, J ) 8.1 Hz, H-6
tolyl), 6.74 (1H, t, J ) 5.9 Hz, NH), 4.95 (2H, s, CH2O), 3.95 (3H, s,
OCH3), 3.30 (2H, q, J ) 6.9 Hz, H-6), 2.32 (2H, t, J ) 7.4 Hz, H-2),
2.23 (3H, s, CH3-tolyl), 1.63 (2H, quint, J ) 7.4 Hz, H-3), 1.51 (2H,
quint, J ) 7.3 Hz, H-5), 1.35 (2H, m, H-4); 13C NMR (CDCl3), δ
178.91 (C-1), 162.14 (CONH), 156.65 (C-1 tolyl), 151.05 (CdN),
135.78 (C-2 Ph), 130.60 (C-3 tolyl), 129.45 (C-5 Ph), 128.97 (C-3 Ph),
128.70 (C-1 Ph), 127.64 (C-6 Ph), 127.41 (C-4 Ph), 126.84 (C-2 tolyl),
126.72 (C-4 Ph), 120.48 (C-4 tolyl), 111.10 (C-6 tolyl), 68.25 (CH2O),
63.26 (CH3O), 39.29 (C-6), 33.69 (C-2), 29.14 (C-5), 26.25 (C-4), 24.22
(C-3), 16.24 (CH3-Tol); IR (film), 3415, 3065, 2938, 2858, 2809, 1739,
1708, 1670, 1602, 1525, 1495, 1241, 1049, 752 cm-1; MS (EI), m/z
(%) 382 (5), 381 (18), 304 (17), 305 (89), 274 (20), 224 (13), 115
(19), 116 (100); HRMS (FAB), calcd for C23H29N2O5 (M+ + 1)
413.20765, found 413.20673; UV (100 mM sodium phosphate buffer,
pH 7.4), ꢀ (280 nm) ) 2.03 mM-1 cm-1, ꢀ (260 nm) ) 4.62 mM-1
nm) ) 1.55 mM-1 cm-1, ꢀ (260 nm) ) 4.69 mM-1 cm-1
.
(E)-5,9-Dioxo-4-(2-(o-tolyloxymethyl)phenyl)-2-oxa-3,6,10-triaza-
dodec-3-en-12-oic Acid (KMꢀag). Purification of the crude product
obtained from 100 mg of KM0 by column chromatography, eluting
with CHCl3/methanol 99:1, afforded 69 mg of KMꢀag (48% yield).
The compound was a white solid: mp 183–185 °C (from CHCl3/
1
hexane); H NMR (DMSO-d6), δ 12.48 (1H, br s, COOH), 8.26 (1H,
t, J ) 5.8 Hz, C5-NH), 8.20 (1H, t, J ) 5.7 Hz, C9-NH), 7.52 (1H, br
d, J ) 7.5 Hz, H-6 Ph), 7.42 (1H, ddd, J ) 7.5, 7.5, 1.5 Hz, H-5 Ph),
7.36 (1H, ddd, J ) 7.5, 7.5, 1.5 Hz, H-4 Ph), 7.17 (1H, dd, J ) 7.5,
1.4 Hz, H-3 Ph), 7.12 (1H, br d J ) 7.5 Hz, H-3 tolyl), 7.09 (1H, br
t, J ) 8 Hz, H-5 tolyl), 6.86 (1H, br d, J ) 8 Hz, H-6 tolyl), 6.81 (1H,
dd, J ) 7.5 and 7.5 Hz, H-4 tolyl), 4.91 (2H, s, CH2O), 3.88 (3H, s,
OCH3), 3.73 (2H, d J ) 5.7 Hz, H-11), 3.36 (2H, overlapped with
water signal from DMSO-d6, H-7), 2.35 (2H, t J ) 7.2 Hz, H-8), 2.16
(3H, s, CH3-tolyl); 13C NMR (DMSO-d6), δ 171.20 (C-12), 170.72
(C-9), 161.86 (C-5), 156.00 (C-1 tolyl), 151.06 (C-4), 135.30 (C-2 Ph),
130.32 (C-3 tolyl), 129.58 (C-1 Ph), 128.87 (C-5 Ph), 128.54 (C-3 Ph),
127.11 (C-4 Ph and C-6 Ph), 126.74 (C-5 tolyl), 125.82 (C-2 tolyl),
120.25 (C-4 tolyl), 111.11 (C-6 tolyl), 67.27 (CH2O), 62.58 (CH3O),
40.48 (C-11), 35.48 (C-7), 34.44 (C-8), 15.86 (CH3); IR (KBr), 3365,
3290, 3081, 2934, 1755, 1651, 1623, 1568, 1495, 1231, 1050, 1002,
754 cm-1; MS (EI), m/z (%) 396 (6), 320 (18), 223 (28), 191 (7), 144
(3), 116 (100); HRMS (FAB), calcd for C22H26N3O6 (M+ + H)
428.18216, found 428.18244; UV (100 mM sodium phosphate buffer
pH 7.4): ꢀ (280 nm) ) 1.70 mM-1 cm-1, ꢀ (260 nm) ) 5.28 mM-1
cm-1
.
(E)-4–2-(Methoxyimino)-2-(2-(o-tolyloxymethyl)phenyl)acet-
amido)butanoic Acid (KM4). The crude coupling product obtained
from 2.0 g of KM0 was first purified by column chromatography,
eluting with CHCl3/EtOAc 8:2, and then recrystallized from EtOAc to
afford 870 mg of pure KM4 (34% yield) as a white solid: mp 132–134
cm-1
.
1
°C (from benzene/hexane); H NMR (CDCl3), δ 7.55 (1H, br d, J )
(E)-5,8-Dioxo-4-(2-(o-tolyloxymethyl)phenyl)-2-oxa-3,6,9-triaza-
tridec-3-en-13-oic Acid (KMgab). Purification of the crude coupling
product obtained from 132 mg of KM0 by column chromatography,
eluting with CHCl3/methanol 9:1, afforded 86 mg of pure KMgab (44%
yield) as a white solid: mp 170–172 °C (from CHCl3/hexane); 1H NMR
(DMSO-d6), δ 12.04 (1H, br s, COOH), 8.31 (1H, t, J ) 5.7 Hz, C5-
NH), 7.95 (1H, t, J ) 5.5 Hz, C9-NH), 7.55 (1H, dd, J ) 7.5, 1.5 Hz,
H-6 Ph), 7.43 (1H, ddd, J ) 7.5, 7.5, 1.5 Hz, H-5 Ph), 7.38 (1H, ddd,
J ) 7.5, 7.5, 1.5 Hz, H-4 Ph), 7.20 (1H, dd, J ) 7.5, 1.5 Hz, H-3 Ph),
7.11 (1H, br d J ) 7.2 Hz, H-3 tolyl), 7.09 (1H, br t, J ) 7.9 Hz, H-5
tolyl), 6.89 (1H, br d, J ) 7.9 Hz, H-6 tolyl), 6.82 (1H, dd, J ) 7.4
and 7.4 Hz, H-4 tolyl), 4.92 (2H, s, CH2O), 3.92 (3H, s, OCH3), 3.74
(2H, d J ) 5.8 Hz, H-7), 3.07 (2H, q, J ) 6.8 Hz, H-10), 2.21 (2H, t
J ) 7.4 Hz, H-12), 1.62 (2H, quint, J ) 7.2 H-11); 13C NMR (DMSO-
d6), δ 174.13 (C-13), 168.04 (C-8), 162.10 (C-5), 156.02 (C-1 tolyl),
150.71 (C-4), 135.40 (C-2 Ph), 130.34 (C-3 tolyl), 129.40 (C-1 Ph),
128.97 (C-5 Ph), 128.67 (C-3 Ph), 127.15 (C-6 Ph), 127.09 (C-4 Ph),
126.78 (C-5 tolyl), 125.79 (C-2 tolyl), 120.28 (C-4 tolyl), 111.24 (C-6
tolyl), 67.22 (CH2O), 62.75 (CH3O), 41.25 (C-7), 37.85 (C-10), 30.88
(C-12), 24.23 (C-11), 15.84 (CH3); IR (KBr), 3408, 3323, 2937, 1730,
1652, 1568, 1523, 1417, 1240, 1168, 1044, 1014, 747 cm-1; MS (EI),
m/z (%) 441 (M+, 4), 410 (12), 335 (21), 334 (100), 216 (6), 303
(13), 224 (11), 203 (14), 173 (18); HRMS, calcd for C23H27N3O6
441.19998, found 441.18935; UV (100 mM sodium phosphate buffer,
pH 7.4), ꢀ (280 nm) ) 1.66 mM-1 cm-1, ꢀ (260 nm) ) 4.86 mM-1
7.4 Hz, H-6 Ph), 7.43 (1H, ddd, J ) 7.4, 7.4, 1.6 Hz, H-5 Ph), 7.38
(1H, ddd, J ) 7.3, 7.3, 1.4 Hz, H-4 Ph), 7.22 (1H, dd, J ) 7.3, 1.6 Hz,
H-3 Ph), 7.15–7.08 (2H, m, overlapped H-3 tolyl and H-5 tolyl), 6.93
(1H, t, J ) 6.0 Hz, NH), 6.85 (1H, dd, J ) 7.3 and 7.3 Hz, H-4 tolyl),
6.79 (1H, br d, J ) 8.1 Hz, H-6 tolyl), 4.96 (2H, s, CH2O), 3.95 (3H,
s, OCH3), 3.37 (2H, q, J ) 6.7 Hz, H-4), 2.36 (2H, t, J ) 7.1 Hz,
H-2), 2.24 (3H, s, CH3-tolyl), 1.84 (2H, quint, J ) 7.0 Hz, H-3); 13C
NMR (CDCl3), δ 177.95 (C-1), 162.50 (CONH), 156.60 (C-1 tolyl),
150.81 (CdN), 135.81 (C-2 Ph), 130.61 (C-3 tolyl), 129.49 (C-5 Ph),
128.99 (C-3 Ph), 128.51 (C-1 Ph), 127.63 (C-6 Ph), 127.41 (C-4 Ph),
126.79 (C-2 tolyl), 126.73 (C-4 Ph), 120.51 (C-4 tolyl), 111.10 (C-6
tolyl), 68.23 (CH2O), 63.32 (CH3O), 38.74 (C-4), 31.29 (C-2), 24.49
(C-3), 16.23 (CH3-tolyl); IR (KBr), 3386, 2979, 2939, 2900, 1731, 1629,
1603, 1540, 1496, 1247, 1184, 1013, 749 cm-1; MS (EI), m/z (%) 354
(2), 353 (7), 277 (39), 259 (3), 246 (8), 224 (7), 223 (11), 192 (3), 144
(3), 117 (17), 116 (100); HRMS (FAB), calcd for C21H25N2O5 (M+
+
1) 385.17635, found 385.17714; UV (100 mM sodium phosphate buffer,
pH 7.4), ꢀ (280 nm) ) 2.19 mM-1 cm-1, ꢀ (260 nm) ) 4.88 mM-1
cm-1
.
(E)-5,8-Dioxo-4-(2-(o-tolyloxymethyl)phenyl)-2-oxa-3,6,9-triaza-
undec-3-en-11-oic Acid (KMgg). Purification of the crude coupling
product obtained from 183 mg of KM0 by column chromatography,
eluting with CHCl3 and then CHCl3/EtOAc 1:1, afforded 116 mg of
pure KMgg (46% yield) as a white solid: mp 129–131 °C (from CHCl3/
1
hexane); H NMR (DMSO-d6), δ 12.60 (1H, br s, COOH), 8.39 (1H,
cm-1
.
t, J ) 5.8 Hz, C5-NH), 8.26 (1H, t, J ) 5.8 Hz, C8-NH), 7.54 (1H, br
d, J ) 7.5 Hz, H-6 Ph), 7.41 (1H, ddd, J ) 7.5, 7.5, 1.5 Hz, H-5 Ph),
Preparation of Protein-Hapten Conjugates. All conjugates used
in this study were prepared by activation of the free carboxylic group

1548 J. Agric. Food Chem., Vol. 56, No. 5, 2008
Mercader et al.
of the hapten and reaction with the amine groups of the carrier protein.
Two carrier proteins were used: BSA for the immunogenic conjugate
and OVA for coating conjugates.
competitor. The ratio of both absorbances was used as the criterion for
selecting antibody-secreting clones. The selected hybridomas were
cloned by limiting dilution in HT cloning medium (s-DMEM containing
20% FBS and supplemented with 100 µM hypoxanthine, 16 µM
thymidine, and 1% HFCS). Stable antibody-producing clones were
expanded and cryopreserved in liquid nitrogen.
Purification of Monoclonal Antibodies. MAbs were purified from
late stationary phase culture supernatants by ammonium sulfate
precipitation and protein G affinity chromatography following the
manufacturer’s instructions. The purified mAbs were stored at 4 °C as
ammonium sulfate precipitates, and the immunoglobulin isotype was
determined using the ImmunoPure Monoclonal Antibody Isotyping Kit
I (HRP/ABTS) from Pierce (Rockford, IL).
Immunogenic Conjugate. Fifty micromoles of KM6 was dissolved
in 500 µL of DMF and mixed with 50 µmol of NHS and 50 µmol of
DCC also in DMF. Additional DMF was added to bring the final
concentration of all reagents to 50 mM. The hapten was activated
overnight at room temperature in amber vials. The day after, the reaction
was centrifuged and the supernatant was collected. Next, 400 µL of
the activated hapten solution was added dropwise to 2 mL of a 15
mg/mL BSA solution in 50 mM carbonate-bicarbonate buffer, pH 9.6.
The coupling reaction was allowed during 4 h at room temperature
with moderate stirring. The initial hapten-to-protein molar ratio in the
mixture was aproximately 40:1. Finally, the conjugate was separated
from the uncoupled hapten by gel filtration on Sephadex G-25, using
100 mM phosphate buffer, pH 7.4, as eluant. The degree of hapten
conjugation to protein was measured spectrophotometrically. If con-
jugation occurred, the UV–vis spectrum of the conjugate was slightly
different from that of the free protein. Therefore, the hapten-to-protein
molar ratio was the average calculated from the absorbance values at
280 and 260 nm by assuming that the molar absorption of the haptens
and the protein were the same for the free and the conjugated forms.
The purified conjugate was stored at -20 °C.
Assay Conjugates. The mixed anhydride method was used to prepare
conjugates of each hapten to OVA. Briefly, 18 µmol of hapten was
dissolved in 180 µL of DMF and mixed with 18 µmol of tributylamine
and 18 µmol of isobutyl chloroformate also in DMF. The same solvent
was added to bring the final concentration of all reagents to 90 mM.
Haptens were activated during 1 h at room temperature. Next, 100 µL
of activated hapten solution was added dropwise to 2 mL of a 15 mg/
mL OVA solution in 50 mM carbonate-bicarbonate buffer, pH 9.6.
The coupling reaction was allowed during 2.5 h at room temperature
with moderate stirring. The initial hapten-to-protein molar ratios in the
mixture were approximately 15:1. Finally, conjugates were separated
from uncoupled haptens by gel chromatography as described for the
immunogenic conjugates.
ELISAs. General Procedure for Immunoassays. Ninety-six-well
polystyrene ELISA plates were coated with 100 µL/well of OVA-conju-
gatesolutions(at1.0,0.1,or0.01µg/mL)in50mMcarbonate-bicarbonate
buffer, pH 9.6, by overnight incubation at room temperature. The coated
plates were washed four times with washing solution [0.15 M NaCl
containing 0.05% (v/v) Tween 20] and then received 50 µL/well of
analyte in PBS plus 50 µL/well of hybridoma supernatant or immu-
noglobulin solution in PBS containing 0.05% Tween 20 (PBST), unless
otherwise stated. All samples were run in duplicate wells. Immunologi-
cal reaction took place (typically 2 h) at room temperature, and plates
were washed again as described. Next, 100 µL/well of a 1/2000 dilution
(unless otherwise stated) of RAM-HRP conjugate in PBST was added,
and plates were incubated (typically 1 h) at room temperature. After
washing, retained peroxidase activity was determined by adding 100
µL/well of freshly prepared 2 mg/mL OPD and 0.012% (v/v) H2O2 in
25 mM citrate and 62 mM sodium phosphate buffer, pH 5.4. The
enzymatic reaction was stopped after 10 min at room temperature by
adding 100 µL/well of 2.5 M sulfuric acid. The absorbance was
immediately read at 492 nm with a reference wavelength at 650 nm.
NoncompetitiVe Conjugate-Coated Assays. Purified mAbs were
assessed by bidimensional checkerboard titration versus all coating
conjugates. The stated general conditions were followed, but buffer
containing no analyte was used. Immunoreagent titrations for final assay
development were performed under the specific conditions of each
assay. The optimum mAb concentration for competitive experiments
was determined in each case, for a fixed conjugate concentration, to
reach a maximum absorbance value of 1.0 in the absence of analyte.
CompetitiVe Two-Step Conjugate-Coated ELISAs. These experiments
were carried out according to the general assay conditions previously
described. Assay sensitivity (IC50) was estimated as the concentration
of analyte that reduced 50% the maximum signal (Amax) reached at the
zero dose of analyte. The limit of detection (LOD) was estimated as
the concentration of KM that provided a 10% reduction of Amax. Cross-
reactivity (CR) values were calculated according to the following
formula: CR ) [IC50(KM)/IC50(compound)] × 100.
Production of Monoclonal Antibodies. Animal manipulation has
been performed in compliance with the laws and guidelines of the
Spanish Ministry of Agriculture, Fisheries, and Food.
Immunization. BALB/c female mice (8–10 weeks old) were im-
munized with the BSA-KM6 conjugate by intraperitoneal injections.
Doses consisted of an emulsion of 100 µL of PBS (10 mM phosphate
buffer, pH 7.4, with 137 mM NaCl, 2.7 mM KCl) containing 100 µg
of protein conjugate, estimated as protein concentration, and 100 µL
of Freund’s adjuvants. The first dose contained complete Freund’s
adjuvant, and subsequent doses were given at weeks 3 and 6 using
incomplete Freund’s adjuvant. After a resting period of at least 3 weeks
from the last injection with adjuvant, mice received a booster
intraperitoneal injection of 100 µg of protein conjugate in 200 µL of
PBS, 4 days before cell fusion. Mice were tail-bled 9 or 10 days after
the third injection.
Cell Fusion and Culture. P3-X63/Ag 8.653 murine myeloma cells
were cultured in high-glucose DMEM supplemented with 2 mM alanyl-
glutamine, 1 mM MEM nonessential amino acids, and 25 µg/mL
gentamicin (referred to as s-DMEM) with 10% (v/v) FBS. Just before
spleen extraction, mouse blood was collected by heart puncture and
serum diluted 1/50 in PBS containing 0.01% (w/v) thimerosal and stored
at 4 °C. Cell fusion procedures were carried out essentially as described
by Nowinski et al. (17). Mouse spleen lymphocytes were fused with
myeloma cells at a 4:1 ratio using PEG 1500 as the fusing agent. The
fused cells were distributed in 96-well culture plates at an approximate
density of 2.5 × 105 cells/well in 100 µL of s-DMEM with 20% FBS.
Twenty-four hours after plating, 100 µL of HAT selection medium
(s-DMEM supplemented with 100 µM hypoxanthine, 0.4 µM aminop-
terine, and 16 µM thymidine) with 15% FBS and 1% (v/v) HFCS was
added to each well.
CompetitiVe One-Step Conjugate-Coated ELISA. For this assay only
OVA-KMgab was used as coating conjugate. The general procedure
was followed except for the competitive reaction, for which 50 µL/
well of standard solution in PBS and 50 µL/well of a 1:1 (v/v) mixture
of mAb and RAM-HRP conjugate solutions in PBST were added.
The reaction was allowed to happen for 1 h at room temperature and,
after washing, retained peroxidase activity was revealed.
Stocks and Standard CurVes. Analytes were prepared as concentrated
solutions in DMF and kept at -20 °C in amber glass vials. For standard
curves, a 1/1000 dilution was prepared in PBS from the stock in DMF,
and then it was serially diluted in PBS, always using borosilicate glass
tubes. Competitive curves were obtained by plotting mean absorbance
values versus the logarithm of analyte concentration. Sigmoidal curves
were mathematically fitted to a four-parameter logistic equation using
SigmaPlot software package from SPSS Inc. (Chicago, IL).
RESULTS AND DISCUSSION
Hapten Synthesis and Protein Conjugates. Derivatization
sites opposite to the characteristic chemical groups of a hapten
are usually preferred to obtain highly specific mAbs. Also, to
obtain high-affinity antibodies, it is advisable to introduce a
spacer arm of four to six carbons and to preserve the main
Hybridoma Selection and Cloning. Twelve days after fusion,
hybridoma culture supernatants were screened by simultaneous indirect
noncompetitive and competitive ELISA with 1.0 and 0.1 µg/mL coating
homologous conjugate. The signal in noncompetitive conditions was
compared with the competitive one when 0.5 µM KM was used as

Immunoassays to Kresoxim-methyl
J. Agric. Food Chem., Vol. 56, No. 5, 2008 1549
Table 1. Results of Competitive Assays for KM Using mAb KM6#22 and
Different Coating Conjugates
sets of haptens resembling the molecule of KM were prepared
with a spacer containing a free carboxyl moiety. One set (KM4
and KM6) was synthesized by reaction of the hydrolyzed ester
of KM (KM0) with a linear amino acid. An amino acid of
different length was used in each case (4-aminobutyric acid or
6-aminohexanoic acid) with the result of a hydrophobic spacer
covalently linked to the molecule of KM through an amide
group. The other set (KMgg, KMꢀag, and KMgab) was prepared
similarly but using a dipeptide instead of a linear amino acid.
In this way, a slight heterology (an amide group) was introduced
inside the spacer arm and between the molecule of KM and the
carrier protein, while maintaining very similar linker lengths
(see Table 1). This straightforward synthesis strategy uses
readily available spacers, and it has let us investigate the
influence of a polar chemical group, such as an amide, on the
performance of monoclonal antibodies in competitive immu-
noassays. Spacer arms prepared with dipeptides, such as ꢀ-Ala-
ꢀ-Ala, or containing an amide group, such as L-Lys-ethylendi-
amine or 6-hydroxycaproyl-ꢀ-Ala, have been previously reported
for the improvement of assay sensitivity or catalytic antibody
production (21–23). Overall, the described synthetic procedure
constitutes a general and new strategy for the direct and simple
synthesis of haptens for methoxyiminoacetate strobilurins.
Finally, two different chemistries were used for the coupling
of haptens to proteins. The calculated hapten to protein molar
ratios were 23 for the BSA conjugate and between 4 and 6 for
all OVA conjugates.
coating OVA
conjugate
conjugate concn
mAb concna
(ng/mL)
IC50 for KMb
(nM)
(µg/mL)
KM0 (0 Å)c
1.00
0.10
0.01
60
80
800
669.0d
27.0
17.7
e
KM4 (7.5 Å)
1.00
0.10
0.01
150
70
100
-
136.7
16.0
KM6 (10.6 Å)
KMgg (8.9 Å)
KMꢀag (10.4 Å)
KMgab (11.9 Å)
1.00
0.10
0.01
40
80
400
-
50.6
23.9
1.00
0.10
0.01
50
50
300
-
40.7
15.0
1.00
0.10
0.01
100
50
150
-
157.7
17.0
1.00
0.10
0.01
100
60
100
-
92.7
14.3
a Values determined by noncompetitive bidimensional checkerboard titration and
affording a maximum absorbacne value around 1.0. b Mean of three independent
determinations (n ) 3). c Sum of the length of the C-C, CO-C, and N-C bonds
in the spacer arm. d n )1. e Not determined.
Production of Monoclonal Antibodies. Five mice were
immunized with BSA-KM6 conjugate, and the immunization
was followed by noncompetitive and competitive assays with
antisera collected after the third injection. Three cell fusions
were performed with lymphocytes from those animals showing
the strongest positive response. After cell fusions, seven different
hybridomas were cloned and stored in liquid nitrogen. Six of
the produced antibodies were of the IgG1 isotype, and one of
them was of the IgG2a isotype; all immunoglobulins contained
the κ light chain. For selection of one mAb for further assay
development, competitive two-step conjugate-coated ELISAs
were performed using purified antibodies, KM as analyte, and
conjugate OVA-KM6 prepared at 1.0 and 0.1 µg/mL (results
not shown). From the obtained inhibition curves, mAb KM6#22
was selected for further studies because it afforded the most
sensitive assay (results not shown).
Two-Step Conjugate-Coated ELISAs. Conjugate Selection.
Competitive assays were performed using all of the OVA
conjugates as coating reagents at different concentrations. In
general, it was observed that reducing any coating conjugate
concentration to 0.01 µg/mL afforded more sensitive assays
(Table 1). In fact, at a concentration of 1.00 µg/mL of any OVA
conjugate, no competitive assays were obtained and, even at
0.1 µg/mL coating conjugate, the observed IC50 values remained
generally high. Interestingly, the use of the lowest coating
concentration (0.01 µg/mL) always resulted in sensitive assays,
even though for some conjugates the required mAb concentra-
tion had to be significantly raised (800 ng/mL for OVA-KM0).
In addition, at low coating concentrations, all heterologous
conjugates afforded slightly lower IC50 values (around 16 nM)
than the homologous conjugate (IC50 ) 23.9 nM). Therefore,
modest reductions of the IC50 values were observed in heter-
ologous assays, which is in agreement with the commonly found
variations when heterologies are used in monoclonal antibody-
based immunoassays (24, 25). At 0.01 µg/mL coating conjugate,
the lowest amount of mAb was required when OVA-KM4 and
OVA-KMgab were used. Although the homologous and all
Figure 3. Chemical structures of some common strobilurin fungicides.
The CR values are indicated in parentheses with respect to the IC50 value
of kresoxim-methyl. They are the mean of three independent determinations.
geometric and electronic properties of the analyte. Thus, the
immunizing hapten was synthesized with a simple, lineal
hydrocarbonated spacer at the toxophore moiety of the strobi-
lurin molecule distal to the characteristic phenoxy-methyl ring
of KM, so minimum changes were introduced in the chemical
structure of the pesticide. Besides, hapten heterology has been
demonstrated as a proper strategy for the improvement of assay
sensitivity (18–20). In the present work, heterologous haptens
with linker-length and linker-composition differences have been
prepared (Figure 2). The hydrolysis of KM afforded, with good
yields (85.6%), the intermediate carboxylate product (KM0) used
for the subsequent reaction with different spacer chains. Two

1550 J. Agric. Food Chem., Vol. 56, No. 5, 2008
Mercader et al.
100 ng/mL and the secondary reaction with the enzyme-
labeled conjugate, at a 1/2000 dilution, was allowed during
1 h. For the rapid assay, the mAb concentration was fixed at
200 ng/mL and detection with the RAM-HRP conjugate, at
a 1/1000 dilution, was performed during 0.5 h. For competi-
tive assays, mAb solutions were prepared in PBS containing
decreasing concentrations of Tween 20. The Amax/IC50 ratios
of the resulting curves were calculated, and these values were
taken as a measure to set the maximum percentage of Tween
20 acceptable in the assay. Figure 4 shows the dependence
of the Amax/IC50 ratio for various concentrations of this
detergent. From this study, it was found that Tween 20 had
an important and negative influence on assay parameters. On
the one hand, for the extended assay, a plateau was reached
for the Amax/IC50 ratio at Tween 20 concentrations below
0.00625%. On the other hand, even the lowest Tween 20
concentration had a detrimental effect to assay parameters
when the rapid assay was used, the highest Amax/IC50 ratio
for the rapid assay being obtained with no detergent in the
assay buffer. Low background signals were obtained for the
extended and rapid assays (below 0.1 and 0.2 absorbance
unit, respectively). Inhibition curves obtained under optimized
immunoreagent and Tween 20 concentrations are shown in
Figure 5, and assay conditions and parameters are sum-
marized in Table 2. Lower IC50 values were observed with
the rapid assay (5.6 nM compared to 9.7 nM for the extended
assay), but an increase of mAb and RAM-HRP concentra-
tions was required and a higher background was obtained.
Under these conditions, the calculated LODs for KM were
0.4 ng/mL for the extended assay and 0.3 ng/mL for the rapid
assay. Therefore, two-step indirect ELISAs with different
incubation times have been developed that could be valid
for different application, the most suitable assay depending
on time, cost, and sensitivity requirements.
One-Step Conjugate-Coated ELISA. Conjugate-coated
ELISAs are usually robust and easy to perform. In addition,
compared to the antibody-immobilized format, conjugate-coated
ELISAs are more tolerant to hapten heterologies for competitive
assays. Nevertheless, it usually takes longer to carry out because
it includes two incubation steps and an additional washing step.
This drawback could be overcome by incubation of mAb,
analyte solution, and labeled antimouse IgG antibody in just
one step. In this respect, the optimum concentrations of mAb
KM6#22 and RAM-HRP were ascertained by noncompetitive
bidimensional checkerboard titration. Solutions of mAb KM6#22
and RAM-HRP in PBST were mixed in a 1:1 ratio (v/v) and
immediately used. Anyhow, no difference in Amax or IC50 values
was observed when the mixture was incubated for up to 30 min
at room temperature before use (results not shown). Next, the
influence of Tween 20 on the assay parameters of this one-step
assay was also studied using 300 ng/mL final mAb concentration
and 1/2000 RAM-HRP final dilution. From results depicted
in Figure 4, it could be concluded that the lower the concentra-
tion of Tween 20, the higher the Amax/IC50 ratio. Nevertheless,
at 0% Tween 20, high unspecific binding was observed and the
experimental results could not be fitted to an inhibition curve
Figure 4. Results of the study on the influence of Tween 20 on the assay
parameters for the two-step extended assay (black bars), the two-step
rapid assay (white bars), and the one-step assay (gray bars). Results
are the mean of three independent determinations.
heterologous conjugates resulted in similar and sensitive assays,
OVA-KMgab was chosen for further assay optimization
because it afforded a low IC50 value (14.3 nM) at low conjugate
and antibody concentrations.
Antibody SelectiVity. Competitive assays were performed to
study antibody selectivity against five strobilurin fungicides:
azoxystrobin, trifloxystrobin, picoxystrobin, pyraclostrobin, and
dimoxystrobin. Assays were carried out with plates coated with
0.01 µg/mL OVA-KMgab, mAb KM6#22 at 200 ng/mL in
PBST, and serial dilutions of each analyte in PBS. Only
dimoxystrobin was also recognized by mAb KM6#22 with a
CR value of 409% with respect to KM (Figure 3). This result
is not surprising because kresoxim-methyl and dimoxystrobin
have very similar chemical structures and the toxophore moiety,
where the spacer arm had been introduced, contains an amide
group in dimoxystrobin, which resembles the amide formed at
the derivatization site upon reaction with the linker. Although
the immunizing conjugate had been prepared with a derivative
of KM distal to the characteristic ring of the fungicide, it seems
that the toxophore moiety is part of the epitope recognized by
the antibody.
Assay Optimization. Detergents are often added to the assay
buffer to improve the solubility of the analyte or to decrease
nonspecific binding. Tween 20 is one of the additives
commonly used. Nevertheless, it has been published before
that this detergent may also reduce the sensitivity of the
assay (26, 27). Therefore, the influence of detergent concen-
tration on the Amax and IC50 values was studied. Assays were
performed using a solution of 0.01 µg/mL OVA-KMgab for
plate coating. Previous to competitive assays, mAb KM6#22
concentrations were established by noncompetitive bidimen-
sional checkerboard titration, wherein the immunoreaction
was carried out during 2.0 (extended assay) or 0.5 h (rapid
assay) in buffer containing 0.025% Tween 20. For the
extended assay, the mAb concentration was established at
Table 2. Summary of Assay Conditions and Sensitivity
time, first
time, second
reaction (h)
mAb concn
(ng/mL)
Tween
20 (%)
enzyme-labeled
LOD for KMa
(ng/mL)
IC50 for KMa
(nM)
assay
reaction (h)
conjugate dilution
two-step extended assay
two-step rapid assay
one-step assay
2
0.5
1
1
0.5
80
150
300
0.00625
0
0.00625
1/2000
1/1000
1/2000
0.4
0.3
0.4
9.7
5.6
9.2
a Values are the mean of three independent determinations.

Immunoassays to Kresoxim-methyl
J. Agric. Food Chem., Vol. 56, No. 5, 2008 1551
(5) Bartlett, D. M.; Clough, J. M.; Godwin, J. R.; Hall, A. A.; Hamer,
M.; Parr-Dobrzanski, B. The strobilurin fungicides. Pest Manag.
Sci. 2002, 58, 649–662.
(6) Pasquer, F.; Isidore, E.; Zarn, J.; Keller, B. Specific patterns of
changes in wheat gene expression after treatment with three
antifungal compounds. Plant Mol. Biol. 2005, 57, 693–707.
(7) EC Health and Consumer Protection D.G. Plant Health. Technical
review reports on new active substancesskresoxim-methyl, 1998.
(8) Gisi, U.; Sierotzki, H.; Cook, A.; McCaffery, A. Mechanisms
influencing the evolution of resistance to Qo inhibitor fungicides.
Pest Manag. Sci. 2002, 58, 859–867.
(9) EU Council Directives 76/895/EEC, 86/362/EEC, and 90/642/
EEC, 2004.
(10) Cabras, P.; Angioni, A.; Garau, V. L.; Pirisi, F. Gas chromato-
graphic determination of azoxystrobin, fluazinam, kresoxim-
methyl, mepanipyrim, and tetraconazole in grapes, must, and wine.
J. AOAC Int. 1998, 81, 1185–1189.
(11) Li, J. Z.; Wu, X.; Hu, J. Y. Determination of fungicide kresoxim-
methyl residues in cucumber and soil by capillary gas chroma-
tography with nitrogen-phosphorus detection. J. EnViron. Sci.
Health, B 2006, 41, 427–436.
(12) De Melo Abreu, S.; Correia, M.; Herbert, P.; Santos, L.; Alves,
A. Screening of grapes and wine for azoxystrobin, kresoxim-
methyl and trifloxystrobin fungicides by HPLC with diode array
detection. Food Addit. Contam. 2005, 22, 549–556.
(13) Knopp, D. Immunoassay development for environmental analysis.
Anal. Bioanal. Chem. 2006, 385, 425–427.
(14) Furzer, G. S.; Veldhuis, L.; Hall, J. C. Development and
comparison of three diagnostic immunoassay formats for the
detection of azoxystrobin. J. Agric. Food Chem. 2006, 54, 688–
693.
(15) Wingert, H.; Sauter, H.; Brand, S.; Wenderoth, B.; Lorenz, G.;
Ammermann, E. Preparation of methyl phenyl(di)thioglyoxylate
O-methyloximes and analogs as agrochemical and industrial
fungicides. Eur. Pat. Appl. EP 432503 A1, 1991; 149 pp.
(16) Banerjee, K.; Ligon, A. P.; Schuermann, M.; Preut, H.; Spiteller,
M. (E,E)-R-(Methoxyimino)-2-[({1-[3-trifluoromethyl)phenyl]ethyl-
idene}amino)oxymethyl]benzeneacetic acid. Acta Crystallogr.
2005, E61 (6), o1528–o1529.
(17) Nowinski, R. C.; Lostrom, M. E.; Tam, M. R.; Stone, M. R.;
Burnette, W. N. The isolation of hybrid cell lines producing
monoclonal antibodies against the p15(E) protein ecotropic murine
leukemia viruses. Virology 1979, 93, 11–126.
(18) Holthues, H.; Pfeifer-Fukumura, U.; Sound, I.; Bauman, W.
Evaluation of the concept of heterology in a monoclonal antibody-
based ELISA utilizing direct hapten linkage to polystyrene
microtiter plates. J. Immunol. Methods 2005, 304, 68–77.
(19) Estevez, M. C.; Kreuzer, M.; Sanchez-Baeza, F.; Marco, M. P.
Analysis of nonylphenol: Advances and improvements in the
immunochemical determination using antibodies raised against
the technical mixture and hydrophilic immunoreagents. EnViron.
Sci. Technol. 2006, 40, 559–568.
Figure 5. Inhibition curves of kresoxim-methyl for the two-step extended
assay (circles), the two-step rapid assay (squares), and the one-step
assay (triangles). Each point is the mean of three independent
determinations.
and no Amax/IC50 ratio could be calculated. Therefore, the lowest
assayed Tween 20 concentration providing a reproducible
inhibition curve was chosen for further assay development.
Optimized mAb, RAM-HRP, and Tween 20 concentrations
are summarized in Table 2. The mean inhibition curve of three
independent experiments is shown in Figure 5. An IC50 value
of 9.2 nM was obtained when the experimental values were
fitted to a sigmoidal curve from which the LOD for KM was
established at 0.4 ng/mL. Thus, a third ELISA has been
developed with reduced washing and incubation steps for the
detection of KM in the low nanomolar range, although a higher
concentration of mAb was required. Similar results were found
by Moreno et al. (28) using the same approach.
To the best of our knowledge, this is the first monoclonal
antibody to KM so far reported in the literature. The obtained
immunoreagents have proved to be suitable for the development
of sensitive strobilurin analytical tests. No large differences in
assay sensitivity were found by the use of linker heterologies.
Three conjugate-coated ELISAs have been optimized using one
or two incubation steps during different incubation periods.
Tween 20 is usually included as additive reagent in buffer, but
our results confirm that a careful optimization of this component
is advisable for proper assay performance. Pending studies
include the performance of dipeptide linkers in immunizing
conjugates and the evaluation of other linker, site, or structure
heterologies for assay development.
(20) Mercader, J. V.; Montoya, A. Development of monoclonal ELISAs
for azinphos-methyl. 1. Hapten synthesis and antibody production.
J. Agric. Food Chem. 1999, 47, 1276–1284.
(21) Hatzidakis, G. I.; Tsatsakis, A. M.; Krambovitis, E. K.; Spyros,
A.; Eremin, S. A. Use of L-lysine fluorescence derivatives as
tracers to enhance the performance of polarization fluoroimmu-
noassays. A study using two herbicides as model antigens. Anal.
Chem. 2002, 74, 2513–2521.
(22) Matsushita, M.; Hoffman, T. Z.; Ashley, J. A.; Zhou, B.; Wirshing,
P.; Janda, K. D. Cocaine catalytic antibodies: the primary
importance of linker effects. Bioorg. Med. Chem. Lett. 2001, 11,
87–90.
(23) Qi, L.; Yamamoto, N.; Meijler, M. M.; Altobell, L. J., III; Koob,
G. F.; Wirsching, P.; Janda, K. D. ∆9-Tetrahydrocannabinol
immunochemical studies: haptens, monoclonal antibodies, and a
convenient synthesis of radiolabeled ∆9-tetrahydrocannabinol.
J. Med. Chem. 2005, 48, 7389–7399.
ACKNOWLEDGMENT
We thank Laura López for excellent technical assistance.
LITERATURE CITED
(1) Anke, T.; Oberwinkler, F.; Steglich, W.; Schramm, G. The
strobilurinssnew antifungal antibiotics from the basidiomycete
Strobilurus tenacellus. J. Antibiot. 1977, 30, 806–810.
(2) Sauter, H.; Steglich, W.; Anke, T. Strobilurins: evolution of a
new class of active substances. Angew. Chem., Int. Ed. 1999, 38,
1328–1349.
(3) Schirmer, U.; Karbach, S.; Pommer, E. H.; Ammermann, E.;
Steghlich, W.; Schwalge, B. A. M.; Anke, T. (BASF); EP 203606
and EP 203608 (priority date, May 30, 1985).
(4) EU Commission Directive 1999/1/EC (1999).

1552 J. Agric. Food Chem., Vol. 56, No. 5, 2008
Mercader et al.
(24) Gui, W. J.; Jin, R. Y.; Chen, Z. L.; Cheng, J. L.; Zhu, G. N.
Hapten synthesis for enzyme-linked immunoassay of the insec-
ticide triazophos. Anal. Biochem. 2006, 357, 9–14.
(28) Moreno, M. J.; Abad, A.; Montoya, A. Production of monoclonal
antibodies to the N-methylcarbamate pesticide propoxur. J. Agric.
Food Chem. 2001, 49, 72–78 .
(25) Kim, Y. J.; Kim, Y. A.; Lee, Y. T.; Lee, H. S. Enzyme-linked
immonosorbent assays for the insecticide fenitrothion. Influence
of hapten conformation and sample matrix on assay performance.
Anal. Chim. Acta 2007, 591, 183–190.
(26) Mercader, J. V.; Montoya, A. Development of monoclonal ELISAs
for azinphos-methyl. 2. Assay optimization and water sample
analysis. J. Agric. Food Chem. 1999, 47, 1285–1293.
(27) Abad, A.; Montoya, A. Development of an enzyme-linked
immunosorbent assay to carbaryl. 2. Assay optimization and
application to the analysis of water samples. J. Agric. Food Chem.
1997, 45, 1495–1501.
Received for review October 16, 2007. Revised manuscript received
December 28, 2007. Accepted December 28, 2007. This work was
supported by Instituto Nacional de Investigación y Tecnología Agraria
y Alimentaria (INIA; Grant CAL03-005) and cofinanced by FEDER
funds. Partial funding by Agroalimed, Conselleria d’Agricultura, Pesca
i Alimentació, is also acknowledged. J.V.M. was hired by the CSIC
under an I3P contract financed by the Spanish Ministry of Education
and the European Social Fund. C.S. acknowledges a research fellowship
from the I3P-CSIC program and the European Social Fund.
JF073039X