Immunoassays for trifloxystrobin analysis. Part I. Rational design of regioisomeric haptens and production of monoclonal antibodies

Article abstract of DOI:10.1016/j.foodchem.2013.11.150

Trifloxystrobin is one of the main active principles belonging to the strobilurin family of crop protection compounds. In this article, the synthesis of a battery of regioisomeric functionalized derivatives of trifloxystrobin is described. The same aliphatic linear carboxylated chain was introduced as spacer arm in all of the synthesized haptens, but it was located at different positions of the parent molecule. N,N′-Disuccinimidyl carbonate was employed for hapten activation, so the resulting N-hydroxysuccinimyl ester could be readily purified and efficiently coupled to proteins. After immunization and hybridoma generation, a collection of 20 mouse monoclonal antibodies from different immunizing haptens was obtained. The analytical performance of these immunoreagents was evaluated in terms of affinity and selectivity with the aim to develop rapid and practical immunochemical procedures for trifloxystrobin determination.

Full text of DOI:10.1016/j.foodchem.2013.11.150

Food Chemistry 152 (2014) 230–236
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Immunoassays for trifloxystrobin analysis. Part I. Rational design
of regioisomeric haptens and production of monoclonal antibodies
Rosario López-Moreno a,1, Josep V. Mercader b,1, Consuelo Agulló a, Antonio Abad-Somovilla a,
,
⇑
Antonio Abad-Fuentes b,
⇑
a Department of Organic Chemistry, Universitat de València, Doctor Moliner 50, 46100 Burjassot, València, Spain
b Institute of Agrochemistry and Food Technology, Consejo Superior de Investigaciones Científicas (IATA–CSIC), Agustí Escardino 7, 46980 Paterna, València, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 August 2013
Received in revised form 20 November 2013
Accepted 23 November 2013
Available online 1 December 2013
Trifloxystrobin is one of the main active principles belonging to the strobilurin family of crop protection
compounds. In this article, the synthesis of a battery of regioisomeric functionalized derivatives of
trifloxystrobin is described. The same aliphatic linear carboxylated chain was introduced as spacer arm
in all of the synthesized haptens, but it was located at different positions of the parent molecule. N,N0-
Disuccinimidyl carbonate was employed for hapten activation, so the resulting N-hydroxysuccinimyl
ester could be readily purified and efficiently coupled to proteins. After immunization and hybridoma
generation, a collection of 20 mouse monoclonal antibodies from different immunizing haptens was
obtained. The analytical performance of these immunoreagents was evaluated in terms of affinity and
selectivity with the aim to develop rapid and practical immunochemical procedures for trifloxystrobin
determination.
Keywords:
Strobilurin
Fungicide
Regioisomeric haptens
Active ester
Ó 2013 Elsevier Ltd. All rights reserved.
Derivatization site
Immune response
1. Introduction
Trifloxystrobin shows low toxicity to mammals, birds, bees,
terrestrial organisms, and some plants, but it is highly noxious to
Strobilurins are a new growing class of synthetic fungicide
compounds that are used for plant protection. During the last five
years, the number of registered strobilurins has doubled, revealing
the great commercial success of these bioactive substances. Now-
adays, one of the best sold strobilurins is trifloxystrobin (Bayer,
2010). Structurally, trifloxystrobin is characterized by a methyl
(E)-2-(methoxyimino)-2-phenylacetate toxophore group and a tri-
fluoromethylphenyl moiety linked by a (E)-ethylideneaminooxym-
ethyl bridge (Fig. 1). Out of the four possible geometric isomers of
the trifloxystrobin molecule (EE, EZ, ZE, and ZZ), the EE isomer is by
far the most effective to combat fungal diseases. Accordingly, just
the EE isomer is used in the commercial fungicide formulations
and therefore it is the only residue sought in common pesticide
monitoring programs (Banerjee, Ligon, & Spiteller, 2005). Like the
other members of this family of fungicides, it is classified as QoI
inhibitor, blocking mitochondrial respiration in fungi of the four
main groups of plant pathogens, i.e., ascomycetes, basidiomycetes,
deuteromycetes, and oomycetes (Balba, 2007; Bartlett et al., 2002).
fishes, aquatic organisms, and algae. Microbial metabolism
primarily consists in hydrolysis of the methyl ester group
(Banerjee, Ligon, & Spiteller, 2006, 2007).
Immunochemical methods constitute alternative approaches
for chemical residue analysis. The successful generation of specific
antibodies and sensitive assays to a small organic molecule, such as
a pesticide, is greatly dependent upon a proper design of immuniz-
ing and assay haptens. Conformation and electronic features of the
target compound should be mimicked by the immunizing hapten,
and a proper display of its main characteristic chemical moieties as
part of a protein conjugate has to be maximized (Mercader, Agulló,
Abad-Somovilla, & Abad-Fuentes, 2011; Shan, Lipton, Gee, &
Hammock, 2003). Consequently, hapten and protein are commonly
linked through a spacer arm, which must show low immunogenic-
ity and must not interfere with antibody binding. Most commonly,
a linear aliphatic chain of 3–6 carbon atoms is employed as linker
(Abad, Moreno, & Montoya, 1998; Kim et al., 2003), though good
results with shorter linkers have been reported (Kong, Zhang,
Zhang, Gee,
& Li, 2010; Suárez-Pantaleón, Mercader, Agulló,
Abad-Somovilla, & Abad-Fuentes, 2008). In a previous article, anti-
bodies to trifloxystrobin were produced from a single immunizing
hapten without spacer arm (Mercader, Suárez-Pantaleón, Agulló,
Abad-Somovilla, & Abad-Fuentes, 2008a). However, trifloxystrobin
⇑
Corresponding authors. Tel.: +34 963544509; fax: +34 963544328
(A. Abad-Somovilla). Tel.: +34 963900022; fax: +34 963636301 (A. Abad-Fuentes).
E-mail addresses: antonio.abad@uv.es (A. Abad-Somovilla), aabad@iata.csic.es
(A. Abad-Fuentes).
is
a very flexible molecule that can adopt many different
1
These authors contributed equally to this work.
0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.11.150

R. López-Moreno et al. / Food Chemistry 152 (2014) 230–236
231
A
B
Fig. 1. (A) Global minimum energy conformation of trifloxystrobin. Calculations were performed using Molecular Mechanics (MM3) as implemented in the CAChe program. A
systematic conformational search was performed (all rotatable bonds were rotated by 15 degree steps) and the geometry of the generated conformers was refined by
performing an optimize geometry calculation in MOPAC using PM3 parameters. This conformer represents the 2.6% of the equilibrium conformer population. Arrows denote
the spacer-arm attachment site in each of the haptens. (B) Structure of trifloxystrobin (TF), the synthesized haptens, and the corresponding N-succinimidyl esters (activated
haptens).
energetically similar conformations – even in the crystalline solid
state, it adopts two different conformations which appear mixed
in the obtained X-ray structure (Ziegler, Benet-Buchholtz, Etzel, &
Gayer, 2003). Due to this mobility, it is not easy to predict, not even
using computer-assisted molecular modeling, which position of
the molecular skeleton would be more suitable for incorporation
of the spacer arm in order to prepare an immunizing conjugate
with prolonged and advantageous display of the hapten to the im-
mune system, thereby increasing the chance of producing antibod-
ies with enhanced binding properties against the target analyte. In
the present study, we report the rational design and synthesis of
seven haptens with the only difference of linker positioning in
the trifloxystrobin framework, thus covering a great variety of pos-
sible spatial orientations of the molecule. The performance of the
prepared regioisomeric molecules as immunogens was evaluated
through the generation of mouse monoclonal antibodies (mAb).
The novel collection of immunoreagents was characterized in the
two main competitive enzyme-linked immunosorbent assay (cEL-
ISA) formats, i.e., direct and indirect, and using homologous and
heterologous conjugates. Moreover, trifloxystrobin structural ana-
logues were synthesized and used as competitors in order to map
epitope molecular interactions. In the following article of this ser-
ies, the best combinations of antibody and conjugate were em-
ployed for immunoassay development, and subsequently for
trifloxystrobin analysis in food samples.
File. Horseradish peroxidase (HRP), ovalbumin (OVA), Freund’s
adjuvants, and o-phenylenediamine were purchased from Sigma/
Aldrich (Madrid, Spain). Bovine serum albumin (BSA) fraction V
was from Roche Applied Science (Mannheim, Germany). Sephadex
G-25 HiTrap Desalting columns for protein–hapten conjugate puri-
fication and Sepharose HiTrap Protein G HP columns for antibody
purification were obtained from GE Healthcare (Uppsala, Sweden).
Rabbit anti-mouse immunoglobulin polyclonal antibody conju-
gated to peroxidase (RAM–HRP) was bought from Dako (Glostrup,
Denmark). Immunoglobulin isotype was determined using the
Mouse MonoAb-ID kit from Invitrogen (Carlsbad, CA, USA). Costar
flat-bottom high-binding 96-well polystyrene ELISA plates were
from Corning (Corning, NY, USA). UV–visible spectra and ELISA
absorbances were read with a PowerWave HT from BioTek Instru-
ments (Winooski, VT, USA). Microwells were washed with an
ELx405 microplate washer also from BioTek Instruments.
2.2. Synthesis of haptens and active esters
Seven functionalized regioisomeric haptens of trifloxystrobin
with equivalent spacer arms located at different positions of the
trifloxystrobin skeleton were synthesized (Fig. 1). Full experimen-
tal details and physical and spectroscopic data of all of the haptens
and synthetic intermediates are provided in Supplementary Data
File (Figs. S1–S6). Carboxylated haptens were activated by forma-
tion of the corresponding N-hydroxysuccinimidyl ester (NHS-
ester) using N,N0-disuccinimidyl carbonate (DSC) and Et3N in dry
acetonitrile as previously described (Esteve-Turrillas et al., 2010).
Briefly, the hapten (0.074 mmol) and DSC (25 mg, 0.096 mmol)
2. Materials and methods
2.1. Chemicals and instrumentation
were dissolved in anhydrous acetonitrile (730
lL) under nitrogen
Analytical grade trifloxystrobin (methyl (E)-methoxyimino-
in an ice-water bath. Et3N (40 L, 0.281 mmol) was then added
l
[(E)-
a
-[1-(
a
,
a
,
a
-trifluoro-m-tolyl)ethylideneaminooxy]-o-
and the resulting mixture was stirred at room temperature until
complete consumption of starting material (as observed by thin-
layer chromatography). The reaction mixture was diluted with
CHCl3, washed with a 10% aqueous solution of NaHCO3 and brine,
and dried over anhydrous Na2SO4. The residue obtained after
tolyl]acetate) was kindly provided by Bayer CropScience
(Frankfurt, Germany). Details about reagents, solvents, synthetic
procedures, and techniques used in the structural characterization
of synthesized compounds are described in Supplementary Data

232
R. López-Moreno et al. / Food Chemistry 152 (2014) 230–236
Table 1
Trifloxystrobin analogues.
evaporation of the solvent was purified by column chromatogra-
phy, using CHCl3 as eluent, affording the NHS-esters in good yield
(60–95%) and in highly pure form, as shown by 1H NMR spectros-
copy (copies of original spectra are included in Supplementary
Data File).
TFa-NHS ester: 86% yield. 1H NMR (300 MHz) d (ppm) 7.87 (1H,
br s, H-200), 7.78 (1H, d, J = 7.8 Hz, H-600), 7.59 (1H, br d, J = 7.8 HZ,
H-400), 7.46 (1H, br t, J = 7.8 Hz, H-500), 7.29 (1H, br s, H-20), 7.20
(1H, dd, J = 7.8, 1.6 Hz, H-60), 7.11 (1H, d, J = 7.8 Hz, H-50), 5.12
(2H, s, OCH2), 4.03 (3H, s, NOCH3), 3.81 (3H, s, CO2CH3), 2.83 (4H,
br s, COCH2CH2CO), 2.67 (2H, t, J = 7.5 Hz, H-6), 2.60 (2H, t,
J = 7.4 Hz, H-2), 2.22 (3H, s, CH3), 1.83–1.65 (4H, m, H-3 and H-
5), 1.49 (2H, m, H-4).
Analogue
R1
R2
R3
TFa-I
TFc-H
TFc-Et
TFf-NO2
TFf-H
CF3
CF3
CF3
NO2
H
CH3
H
C2H5
CH3
CH3
CH3
I
H
H
H
H
H
TFf-NH2
NH2
TFb-NHS ester: 80% yield. 1H NMR (300 MHz) d (ppm) 7.67 (1H,
br s, H-40), 7.59 (1H, br s, H-20), 7.51 (1H, dd, J = 7.6, 1.5 Hz, H-300),
7.46–7.36 (3H, m, H-500, H-60 and H-400), 7.19 (1H, dd, J = 7.4, 1.5 Hz,
H-600), 5.14 (2H, s, OCH2), 4.03 (3H, s, NOCH3), 3.82 (3H, s, CO2CH3),
2.83 (4H, br s, COCH2CH2CO), 2.66 (2H, t, J = 7.3 Hz, H-2), 2.62 (2H,
t, J = 7.4 Hz, H-6), 2.21 (3H, s, CH3), 1.79 and 1.67 (2H each, each m,
H-3 and H-5), 1.47 (2H, m, H-4).
bromo-benzyl derivative. A complete description of the prepara-
tion of these analogues is provided in Supplementary Data File
(Fig. S7).
2.4. Conjugate preparation
TFc-NHS ester: 60% yield. 1H NMR (300 MHz) d (ppm) 7.85 (1H,
br s, H-200), 7.75 (1H, d, J = 7.8 Hz, H-600), 7.59 (1H, d, J = 7.8 Hz, H-
400), 7.51–7.35 (4H, m, H-30, H-40, H-50, and H-500), 7.18 (1H, dd,
J = 7.5, 1.3 Hz, H-60), 5.11 (2H, s, OCH2), 4.03 (3H, s, NOCH3), 3.81
(3H, s, CO2CH3), 2.82 (4H, br s, COCH2CH2CO), 2.75 (2H, t,
J = 7.9 Hz, H-6), 2.55 (2H, t, J = 7.4 Hz, H-2), 1.73 (2H, quint,
J = 7.5 Hz, H-3), 1.56–1.42 (4H, m, H-4 and H-5).
Conjugation was carried out with 1, 5, and 10 lmol of activated
hapten (hapten–NHS ester) and HRP (2.2 mg), OVA (30 mg), and
BSA (15 mg), respectively, in 50 mM carbonate buffer, pH 9.6. For
details see Mercader, Esteve-Turrillas, Agulló, Abad-Somovilla,
and Abad-Fuentes (2012). Protein–hapten conjugates were puri-
fied by gel filtration using 100 mM phosphate buffer, pH 7.4 as elu-
ent, and stored frozen at ꢀ20 °C. Coupling degrees were calculated
at 280 nm from absorbance values before and after conjugation.
TFe-NHS ester: 84% yield. 1H NMR (300 MHz) d (ppm) 7.86 (1H,
br s, H-200), 7.80 (1H, d, J = 7.8 Hz, H-600), 7.59 (1H, d, J = 7.8 Hz, H-
400), 7.48 (1H, m, H-60), 7.46 (1H, m, H-500), 7.42 (1H, m, H-50),
7.37 (1H, m, H-40), 7.18 (1H, dd, J = 7.3, 1.5 Hz, H-30), 5.14 (2H, s,
OCH2), 4.21 (2H, t, J = 6.7 Hz, H-6), 4.02 (3H, s, NOCH3), 2.82 (4H,
br s, COCH2CH2CO), 2.53 (2H, t, J = 7.3 Hz, H-2), 2.23 (3H, s, CH3),
1.73–1.59 (4H, m, H-3 and H-5), 1.44–1.28 (2H, m, H-4).
TFo-NHS ester: 95% yield. 1H NMR (300 MHz) d (ppm) 7.85 (1H,
br s, H-200), 7.79 (1H, d, J = 7.9 Hz, H-600), 7.59 (1H, d, J = 7.8 Hz, H-
400), 7.48 (1H, m, H-60), 7.46 (1H, m, H-500), 7.42 (1H, m, H-50),
7.37 (1H, m, H-40), 7.18 (1H, dd, J = 7.3, 1.5 Hz, H-30), 5.14 (2H, s,
OCH2), 4.25 (2H, t, J = 6.7 Hz, H-6), 3.81 (3H, s, CO2CH3), 2.82 (4H,
br s, COCH2CH2CO), 2.52 (2H, t, J = 7.4 Hz, H-2), 2.22 (3H, s, CH3),
1.68 (4H, m, H-3 and H-5), 1.38 (2H, m, H-4).
2.5. Antibody production
Animal manipulation was carried out in compliance with Span-
ish laws and guidelines (RD1201/2005 and law 32/2007) and
according to European Directive 2010/63EU concerning protection
of animals used for scientific purposes. Each BSA conjugate was
used to immunize a set of female BALB/c mice with 0.1 mg of con-
jugate per animal. Immunogen emulsions were prepared with BSA
conjugates in sterile PBS (10 mM phosphate, pH 7.4 containing
140 mM NaCl) and Freund’s adjuvant. Regular immunization
schedules were followed as previously described (Parra, Mercader,
Agulló, Abad-Somovilla, & Abad-Fuentes, 2012). Ten days after the
third injection, a blood sample was taken by submandibular bleed-
ing. For hybridoma generation, a standard cell fusion protocol was
followed and cells were distributed in 96-well culture microplates
(Mercader et al., 2008a). Selection of the best hybridomas was car-
ried out by a double screening process (Mercader, Suárez-Panta-
león, Agulló, Abad-Somovilla, & Abad-Fuentes, 2008b). Briefly, a
differential ELISA was performed with the supernatant of every
microwell by parallel evaluation of a control well without compet-
itor and a test well with 100 nM trifloxystrobin. Next, those super-
natants exhibiting a clearly lower signal in the test well than in the
control well were reevaluated by a checkerboard competitive
screening test, in which diverse trifloxystrobin concentrations (0,
10, and 100 nM) were tested with serial dilutions of culture super-
natant and different coating concentrations of homologous conju-
gate. Supernatants affording saturated signals in the control well
were also included in this analysis. Hybridomas were cloned twice
by limiting dilution. Antibodies were purified from 100 to 150 mL
culture supernatants by affinity chromatography, and stored at
4 °C as ammonium sulfate precipitates.
TFf-NHS ester: 88% yield. 1H NMR (300 MHz) d (ppm) 7.51 (1H,
dd, J = 7.4, 1.3 Hz, H-300), 7.45–7.34 (4H, m, H-20, H-40, H-400 and H-
500), 7.25 (1H, t, J = 7.7 Hz, H-50), 7.22–7.14 (2H, m, H-600 and H-60),
5.12 (2H, s, OCH2), 4.03 (3H, s, NOCH3), 3.81 (3H, s, CO2CH3), 2.82
(4H, br s, COCH2CH2CO), 2.65–2.58 (2H, two overlapped t,
J = 7.5 Hz, H-2 and H-6), 2.20 (3H, s, CH3), 1.83–1.61 (4H, m, H-3
and H-5), 1.50–1.41 (2H, m, H-4).
TFt-NHS ester: 75% yield. 1H NMR (300 MHz) d (ppm) 7.62 (1H,
t, J = 1.5 Hz, H-20), 7.57–7.47 (2H, m, H-60 and H-300), 7.46–7.33 (3H,
m, H-500, H-400 and H-40), 7.25 (1H, t, J = 7.8 Hz, H-50), 7.18 (1H, dd,
J = 7.4, 1.4 Hz, H-600), 5.12 (2H, s, OCH2), 4.03 (3H, s, NOCH3), 3.82
(3H, s, CO2CH3), 2.84 (4H, br s, COCH2CH2CO), 2.82 (2H, t,
J = 7.4 Hz, H-4), 2.58 (2H, t, J = 7.0 Hz, H-2), 2.18 (3H, s, CH3), 2.06
(2H, quint, J = 7.2 Hz, H-3).
2.3. Synthesis of analogues
A small collection of trifloxystrobin analogues was synthesized,
each one possessing a single structural modification of the triflox-
ystrobin molecule (Table 1). With the exception of TFa-I, an inter-
mediate in the synthesis of hapten TFa, the rest of the analogues
were prepared ex novo from readily available materials via the
same type of strategy used for the preparation of the hapten
framework, which was based on the O-alkylation reaction of a
conveniently functionalized oxime with the appropriated
2.6. Competitive ELISAs
Immunoassays were carried out at room temperature using two
different formats: the antibody-coated direct cELISA and the

R. López-Moreno et al. / Food Chemistry 152 (2014) 230–236
233
conjugate-coated indirect cELISA. Briefly, plates were coated by
overnight incubation at room temperature with immunoreagent
solution in 50 mM carbonate buffer, pH 9.6. For direct assays, coat-
Sonogashira cross-coupling reaction with tert-butyl hex-5-ynoate
and hydrogenation of the triple bond, or by means of an alkylation
reaction with the corresponding tert-butyl bromoester. In all cases,
the synthesis was completed by acid hydrolysis of the tert-butyl
ester moiety.
As illustrated in Fig. 2A, the synthesis of haptens TFa and TFo
implied the O-alkylation reaction of oxime i – prepared according
to the procedure described in the literature by Neufeldt and San-
ford (2010) – and a benzyl bromide such as ii, whose preparation
was carried out from readily available tolyl derivatives following
previously reported methodology (Itoh et al., 1984; Wenderoth
et al., 1990). The incorporation of the C-6 carboxylated spacer
arm was realized by means of an O-alkylation reaction of a hydrox-
yimino group, in the case of hapten TFo, and a palladium catalyzed
Sonogashira cross-coupling reaction followed by hydrogenation of
a triple bond, in the case of hapten TFa.
The synthesis of haptens TFb and TFc involved, as the key step,
the O-alkylation reaction of an oxime such as iii or iv, respectively,
with benzyl bromide v, which was obtained following a modifica-
tion of the literature procedures from commercial 1-(o-tolyl)etha-
none (Hwang, Kim, Kim, & Kyung, 2009; Kim, Kim, Hwang, & Nam,
2009; Wenderoth et al., 1992) (Fig. 2B). For hapten TFb, the prepa-
ration of the intermediate oxime involved bromination of the phe-
nyl ring of 3-(trifluoromethyl)benzoic acid and transformation of
the carboxylic group into a methyl ketone group, followed by
incorporation of the carboxylated alkyl chain through a Sonogash-
ira cross-coupling reaction, hydrogenation of the triple bond, and
condensation of the carbonyl ketone group with hydroxylamine.
On the other hand, the oxime that was needed for the preparation
of hapten TFc was synthesized from 1-(3-(trifluoro-
ing was done with 100
washing the competitive step was performed during 1 h with 50
per well of trifloxystrobin standard solution in PBS and 50 L per
lL per well of antibody solution, and after
lL
l
well of enzyme tracer dilution in PBST (PBS containing 0.05% (v/
v) Tween 20). Indirect competitive assays were developed in OVA
conjugate-coated plates using 50
in PBS and 50 L per well of primary antibody dilution in PBST.
For detection, 100 L per well of RAM–HRP (diluted 1/2000 in
lL per well of analyte solution
l
l
PBST) was used. Both, the competitive and the secondary reaction
were brought to equilibrium by incubation during 1 h. Plates were
washed four times after each step and signal was generated using a
freshly prepared 0.012% (v/v) H2O2 solution in 25 mM citrate and
62 mM phosphate buffer, pH 5.4 containing 2 mg mLꢀ1 o-phenyl-
endiamine. The enzymatic activity was stopped after 10 min with
2.5 M sulfuric acid. Absorbance was read at 492 nm with a refer-
ence wavelength at 650 nm.
2.7. Inhibition curves and data analysis
Starting from a 10 lM trifloxystrobin solution in PBS, standard
curves were prepared by 10-fold serial dilution in the same buffer.
A 10 mM trifloxystrobin stock solution in anhydrous N,N-dimeth-
ylformamide was used to prepare the first standard point. Experi-
mental values were fitted to a four-parameter logistic equation
using the SigmaPlot software package from SPSS Inc. (Chicago, IL,
USA). Antibody affinity was estimated as the trifloxystrobin con-
centration at the midpoint of the sigmoidal curve, usually refers
to as IC50. Cross-reactivity (CR) was calculated as percentage value
from the quotient between the IC50 for trifloxystrobin and the IC50
for the studied analogous compound, both in molar concentration
units.
methyl)phenyl)ethanone through
a-alkylation to the carbonyl
group with tert-butyl 5-bromopentanoate followed by oxime for-
mation. The first transformation did not take place by direct alkyl-
ation of the enolate derived from the methyl ketone group, so it
was necessary to use an indirect alkylation process, based on the
temporal transformation of the methyl ketone group into the cor-
responding allyl b-keto ester derivative (Paulvannan & Chen, 1999;
Snider & Buckman, 1992).
3. Results and discussion
3.1. Preparation of haptens and active esters
Finally, haptens TFf and TFt were synthesized through the same
synthetic sequence, similar to that described above for hapten TFb,
starting from 1-(3-bromophenyl)ethanone (Fig. 2C). After prepara-
tion of the required key oxime vi, O-alkylation with benzyl bro-
mide iv and acid hydrolysis of the tert-butyl ester led to hapten
TFt. On the other hand, hydrogenation of the triple bond previously
to hydrolysis of the tert-butyl ester group afforded hapten TFf.
In order to allow coupling to carrier proteins, the carboxylic
acid group of the haptens was activated through formation of the
corresponding N-succinimidyl ester (see Fig. 1 for structure of
the corresponding NHS-esters). This was readily performed using
DSC as activating reagent. This activation procedure is highly effi-
cient giving exclusively CO2 and N-hydroxysuccinimide as the only
by-products, which allowed an easy purification of the intended
active esters.
A battery of regioisomeric haptens of trifloxystrobin covering
the entire molecular framework was prepared (Fig. 1B). All of the
haptens maintained the structure and main functional groups of
the parent molecule, introducing the minimum possible conforma-
tional and electronic changes, with the linker located at a variety of
positions. In all cases, the spacer arm was a C-6 saturated hydro-
carbon chain, with the exception of TFt, which was functionalized
through the same attachment site that TFf and was synthesized to
assess whether the introduction of a triple bond in the linker may
contribute, through rigidity effects, to an improved hapten
exposure.
Hapten TFe was prepared directly from trifloxystrobin in three
steps via hydrolysis of the methyl ester moiety followed by O-
alkylation of the carboxylate group with tert-butyl 6-bromohex-
anoate and acid hydrolysis of the tert-butyl ester group (see
Fig. S4 and experimental details in Supplementary Data file). The
rest of the haptens were obtained by total synthesis from commer-
cially available starting materials. The synthetic strategy that was
followed for their preparation was based on the Williamson-type
O-alkylation reaction of a conveniently functionalized oxime,
which provided the (1-oxyiminoalkyl)phenyl substructural moiety
(left-hand side of the hapten planar structure in Fig. 1B), with the
appropriate benzyl bromide, which supplied the methyl 2-(alk-
oxyimino)-2-(o-tolyl)acetate subunit (right-hand side of the struc-
ture in Fig. 1B). Incorporation of the alkyl C-6 carboxylated spacer
arm was accomplished either through a palladium catalyzed
3.2. Preparation of hapten–protein conjugates
The availability of the activated haptens in the purified form al-
lowed us to readily prepare both the immunizing and the assay
conjugates using the same coupling procedure and with a good
control of the hapten-to-protein molar ratio (MR). A set of BSA,
OVA, and HRP conjugates for every hapten was obtained with high
yields. Calculated hapten-to-protein MRs were between 8 and 22, 3
and 5, and 3 and 7 for BSA, OVA, and HRP conjugates, respectively.
MRs of BSA and OVA conjugates were in the usual range, whereas
the unlikely estimated values that were observed with some of the

234
R. López-Moreno et al. / Food Chemistry 152 (2014) 230–236
A
B
C
Fig. 2. Schematic diagram showing the rational design and general strategy followed for hapten synthesis.
Table 2
HRP conjugates were probably due to modified molar extinction
coefficients of the protein and/or the hapten after conjugation.
IC50 values (nM) from checkerboard assays using the indirect cELISA format.a
mAb
OVA–hapten coating conjugateb
TFa
TFb
TFc
TFe
TFo
TFf
TFt
3.3. Antibody affinity
c
TFa#11
TFa#24
TFa#26
TFb#14
TFb#32
TFc#146
TFc#151
TFc#163
TFe#23
TFe#25
TFe#38
TFe#310
TFo#14
TFo#110
TFo#112
TFf#13
20.5
11.7
29.9
–
–
–
–
–
–
–
–
–
3.8
12.1
–
–
–
–
1.4
–
–
–
–
–
–
20.7
8.2
17.4
10.2
–
–
–
–
–
–
–
2.7
–
–
–
12.2
–
3.0
–
–
–
–
25.1
2.1
–
–
–
–
2.3
15.4
–
–
–
–
–
–
–
–
–
–
–
–
–
3.4
–
–
–
–
–
–
–
–
–
–
–
–
–
3.2
16.7
–
–
–
–
–
–
–
–
–
–
3.5
–
–
1.8
–
A collection of mAbs to trifloxystrobin was generated using BSA
conjugates of all of the synthesized regioisomeric haptens. A set of
4 mice was immunized with each conjugate, and positive immune
response was confirmed with a small serum sample that was ob-
tained 10 days after the third injection. Cell fusions were carried
out with all of the mice with mostly high efficiencies for hybrid cell
formation. Spleens were processed individually or several spleens
from the same set of animals were pooled. A summary of cell fu-
sion results can be found in Table S1 of Supplementary Data File.
Immunogen BSA-TFa afforded the lowest number (1.2%) of positive
clones – clones segregating antibodies that recognized the immo-
bilized conjugate, whereas conjugate BSA-TFt gave the highest
amount of positive clones (60.7%). Further work with other ana-
lytes and haptens is required to conclusively ascribe this high ratio
of positive clones to the presence of a triple bond in the spacer arm
of hapten Tft. The employed double screening process using a dif-
ferential and a checkerboard approach, as previously described
(Mercader et al., 2008b), allowed identification of those clones pro-
ducing antibodies that bound free trifloxystrobin with high affin-
–
–
–
10.1
6.1
4.2
1.8
22.6
8.5
9.3
–
2.4
–
–
2.4
4.5
3.1
6.7
4.1
1.8
23.1
5.5
11.6
8.0
–
2.9
5.1
15.1
3.8
3.4
5.1
4.4
15.2
5.3
3.3
TFt#21
TFt#35
TFt#211
TFt#316
4.0
–
–
–
–
1.6
a
b
c
Average of three independent experiments.
Conjugate concentration was 1.0
g mLꢀ1
No or low signal at the highest assayed antibody concentration (1.0
l
.
l
g mLꢀ1).
ity. Following that process,
a total of 20 hybridomas were
generated and stabilized – at least two hybridomas were cloned
from each set of immunized animals. All antibodies were consti-
Interestingly, no TFe- or TFo-type antibody bound to conjugates
of TFf or TFt, whereas several TFf- or TFt-type antibodies recog-
nized OVA–TFe or OVA–TFo, or both of them. As a result, it could
be stated that antibodies with the highest affinity were mainly ob-
tained from haptens containing the spacer arm either at the meth-
oxyacetate moiety (haptens TFe and TFo) or at the opposite side of
the molecule (haptens TFb, TFf, and TFt).
Concerning direct assays, the two TFb-type mAbs and most of
the TFa-, TFc-, and TFt-type antibodies did not properly bind any
enzyme tracer. Poor recognition of enzyme tracers by mAbs di-
rectly immobilized to ELISA plates has been previously observed
with other immunoreagents (Abad et al., 1997; Butler, 2000;
tuted by
j light chains and were of the IgG1 isotype, except
antibodies TFt#35 and TFt#316 which were of the IgG2b isotype.
Checkerboard competitive assays were performed by the indi-
rect and direct cELISA formats. Regarding indirect assays, all anti-
bodies recognized the homologous OVA conjugate, and most of
them also afforded enough signal with one or more heterologous
coating antigens (Table 2). IC50 values below 5 nM were obtained
with most types of antibodies, except with those from haptens
with the linker at a more central position of the molecule (TFa
and TFc). Moreover, the mAbs recognized conjugates with the lin-
ker at equivalent positions, such as those of haptens TFe and TFo.

R. López-Moreno et al. / Food Chemistry 152 (2014) 230–236
235
Manclús et al., 2004). Although the tracer recognition capacity of
TFf-NO2
TFf-H
TFf-NH2
several anti-trifloxystrobin antibodies clearly improved when they
were assayed in capture-antibody pre-coated plates (data not
shown), this alternative assay format was not further explored
with those mAbs. All of the TFe- and TFo-type antibodies afforded
enough signal with both TFe and TFo tracers (Table 3), and a similar
behaviour was observed between TFf- and TFt-type mAbs and trac-
ers of TFt and TFf, respectively. Hapten heterology with this format
afforded IC50 values to trifloxystrobin lower than the homologous
assays with monoclonal TFf#13 combined with tracer HRP–TFc,
and with mAb TFt#316 together with HRP–TFe.
100
10
1
3.4. Antibody specificity to structural analogues
0.1
The main structural motifs of the target analyte involved in
antibody binding as a function of the immunizing hapten was stud-
ied by homologous indirect cELISA using as competitors a small
collection of trifloxystrobin analogues carrying single structural
differences that were designed to introduce steric and electronic
modifications in the molecule (Table 1). Interestingly, high CR val-
ues were found (between 100% and 250%) with the iodinated ana-
logue (TFa-I), independently of the antibody type. Equivalent or
even superior recognition of iodinated analogues has been ob-
served in our group with other strobilurins (unpublished results)
and by other authors with brominated compounds (Sanvicens,
TFa
TFb
TFc
TFe
TFo
TFf / t
mAb-type
Fig. 3. Average CR values for three trifloxystrobin analogues obtained with the
different types of antibodies.
properties of the trifluoromethyl group (TFf-NO2) was also well
recognized by the other sorts of antibodies, particularly by TFb-
and TFc-type mAbs – those generated from immunizing haptens
with the spacer arm closer to the linker position of TFf. On the con-
trary, the presence of an electron donating group, such as –NH2
(TFf-NH2), drastically reduced the CR with TFa-, TFb-, TFc-, TFe-,
and TFo-type antibodies, which evidenced the relevance, for anti-
body binding, of the electronic modifications introduced by those
moieties located opposite to the spacer arm of the immunizing
hapten. Binding to an analogue with a neutral substituent (TFf-H)
brought about an intermediate outcome.
Varela,
& Marco, 2003; Suárez-Pantaleón, Mercader, Agulló,
Abad-Somovilla, & Abad-Fuentes, 2011). Most probably, the in-
creased hydrophobicity would compensate the larger size of the
molecule. On the other hand, analogues with slight modifications
at a central position of the molecule, such as TFc-H and TFc-Et,
showed varied CR values (between 10% and 100%) independently
of the antibody origin, even though TFc-type antibodies, derived
from the hapten with the linker at that position, coherently dis-
played the highest recognition of these two analogues (results
not shown).
4. Conclusions
A battery of regioisomeric haptens was prepared with the same
spacer arm – a C-6 hydrocarbon chain ending in a carboxylate
group – located at a variety of positions in order to cover the entire
trifloxystrobin skeleton. In most cases, haptens were synthesized
through formation of a C–C bond using Sonogashira cross-coupling
methodology or C-alkylation reactions, whereas in other cases for-
mation of a C–O bond using an O-alkylation reaction was followed.
Carboxylate activation with DSC and purification of the active ester
was shown to be an attractive strategy for immunogen and assay
conjugate preparation by a single efficient procedure. A key influ-
ence of linker positioning over antibody affinity and specificity
was revealed. For this agrochemical, derivatization over the
methoxyacetate group – an aliphatic peripheral moiety with
high-conformational freedom and displaying the aromatic and
more rigid parts of the molecule – was shown to be the most ade-
quate approach in terms of both synthetic convenience and anti-
body production. Several mAb/conjugate combinations were
shown to be good candidates for immunoassay development with
suitable analytical properties.
Finally, when only the electron withdrawing trifluoromethyl
group of trifloxystrobin was changed, remarkable results were
found. As shown in Fig. 3, average CR values were near 100% for
TFf- and TFt-type antibodies, independently of the modification
that was introduced, as could be predicted by Landsteiner’s princi-
ple, i.e. antibody specificity is directed primarily to the portion of
the hapten located furthest from or opposite to the functional
group linking it to the carrier protein (Landsteiner, 1962). Besides,
the analogue better mimicking the electron withdrawing
Table 3
IC50 values (nM) from checkerboard competitive assay using the direct cELISA
format.a
mAbb
HRP–hapten tracer conjugate
TFa
TFb
TFc
TFe
TFo
TFf
TFt
c
TFa#24
TFc#163
TFe#23
TFe#25
TFe#38
TFe#310
TFo#14
TFo#110
TFo#112
TFf#13
45.1
–
–
–
–
–
–
–
–
–
–
7.9
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
15.9
6.6
6.5
4.5
21.9
6.5
7.5
6.2
–
39.9
5.0
5.7
4.8
–
8.5
7.3
7.3
–
Acknowledgements
This work was supported by the Spanish Ministerio de Ciencia e
Innovación
(MICINN)
(AGL2006-12750-C02-01/02/ALI
and
–
–
AGL2009-12940-C02-01/02/ALI) and cofinanced by FEDER funds.
R.L.-M. was hired by MICINN under a predoctoral FPI grant associ-
ated to the above project. J.V.M. was hired by CSIC with a postdoc-
toral contract under the Ramón y Cajal program, cofinanced by
MICINN and by the European Social Fund (ESF). We thank Ana Iz-
quierdo-Gil and Laura López-Sánchez for excellent technical
assistance.
–
–
–
4.6
–
–
8.1
5.3
10.5
5.6
4.3
9.7
TFt#211
TFt#316
4.1
5.2
–
–
a
Only those mAbs that recognized at least one enzyme tracer are shown. Values
are the mean of three independent experiments.
b
Coating antibody concentration was 1.0
l
g mLꢀ1
No or low signal at the highest assayed tracer concentration (300 ng mLꢀ1).
.
c

236
R. López-Moreno et al. / Food Chemistry 152 (2014) 230–236
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Appendix A. Supplementary material
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at
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