Fluxapyroxad Haptens and Antibodies for Highly Sensitive Immunoanalysis of Food Samples

Article abstract of DOI:10.1021/acs.jafc.7b03199

Fluxapyroxad is a new-generation carboxamide fungicide, with residues increasingly being found in food samples. Immunochemical assays have gained acceptance in food quality control as rapid, cost-effective, sensitive, and selective methods for large sample throughput and in situ applications. In the present study, immunoreagents to fluxapyroxad were obtained for the first time, and competitive immunoassays were developed for the sensitive and specific determination of fluxapyroxad residues in food samples. Two carboxyl-functionalized analogues of fluxapyroxad were prepared, and antibodies with IC₅₀ values in the low nanomolar range were generated from both haptens, though a dissimilar response was observed concerning specificity. A robust direct assay was set up, with a calibration curve exhibiting a limit of detection of 0.05 nM (0.02 μg/L). Limits of quantitation of 5 μg/L were obtained for peach, apple, and grape juices using samples diluted in water. The direct immunoassay was also successfully applied to the determination of fluxapyroxad in grapes from in-field treated grapevines.

Full text of DOI:10.1021/acs.jafc.7b03199

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Article
Fluxapyroxad Haptens and Antibodies for Highly
Sensitive Immunoanalysis of Food Samples
Josep Vicent Mercader, Antonio Abad-Somovilla, Consuelo Agulló, and Antonio Abad-Fuentes
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03199 • Publication Date (Web): 04 Oct 2017
Downloaded from http://pubs.acs.org on October 4, 2017
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Journal of Agricultural and Food Chemistry
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Fluxapyroxad Haptens and Antibodies for Highly
Sensitive Immunoanalysis of Food Samples
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†
‡
‡
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Josep V. Mercader , Antonio Abad-Somovilla , Consuelo Agulló , Antonio Abad-
†
, *
Fuentes
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b
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Department of Biotechnology, Institute of Agrochemistry and Food Technology (IATA),
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Spanish National Research Council (CSIC), Agustí Escardino 7, 46980 Paterna, València, Spain
a
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Department of Organic Chemistry, Universitat de València, Doctor Moliner 50, 46100
Burjassot, València, Spain
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* Corresponding author (Tel. +34-963900022; Fax +34-963636301; E-mail: aabad@iata.csic.es).
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Abstract
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Fluxapyroxad is a new-generation carboxamide fungicide, with residues increasingly
being found in food samples. Immunochemical assays have gained acceptance in food
quality control as rapid, cost-effective, sensitive, and selective methods for large
sample throughput and in situ applications. In the present study, immunoreagents to
fluxapyroxad were obtained for the first time, and competitive immunoassays were
developed for the sensitive and specific determination of fluxapyroxad residues in food
samples. Two carboxyl-functionalized analogues of fluxapyroxad were prepared and
antibodies with IC50 values in the low nanomolar range were generated from both
haptens, though a dissimilar response was observed concerning specificity. A robust
direct assay was set up, with a calibration curve exhibiting a limit of detection of 0.05
nM (0.02 µg/L). Limits of quantitation of 5 µg/L were obtained for peach, apple, and
grape juices using samples diluted in water. The direct immunoassay was also
successfully applied to the determination of fluxapyroxad in grapes from in-field
treated grapevines.
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Keywords
Fungicide, ELISA, antibody, food safety, chemical residue
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INTRODUCTION
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Fluxapyroxad is a new-generation broad-spectrum fungicide that belongs to the
pyrazole carboxamide class of active biocides. It stunts spore germination as well as
germ tube and mycelium growth in the target species via inhibition of the succinate
dehydrogenase (SDH) activity in complex II of the mitochondrial respiratory chain,
which catalyses the oxidation of succinate to fumarate by reducing ubiquinone to
ubiquinol. This enzyme is involved in two essential metabolic pathways, i.e., the
oxidative phosphorylation and the Krebs cycle, which makes SDH an excellent target
for fungi control. This particular and effective mode of action converts SDH inhibitors
into outstanding candidates for co-formulation with other fungicides in order to
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prevent the emergence of resistance. The 2014 global market for SDH-inhibitor
fungicides was valued at $2721 million, and it is expected to grow at a compound
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annual growth rate of 7.1% to mature into a $4046.6 million market by 2020.
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Fluxapyroxad (Figure 1) was recently developed on the basis of the molecular
structure of boscalid, the most successful SDH inhibitor to date, by systematically
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modifying different parts of the molecule. It displays low acute toxicity to humans by
the oral, dermal, and inhalation routes; it is not irritating to the eyes and skin and is
classified as “not likely to be carcinogenic”. The LD50 in rats treated orally or dermally
with fluxapyroxad was greater than 2000 mg/kg of body weight. Low harm to
terrestrial organisms was found; however, acute risk to estuarine/marine species could
occur if formulations of fluxapyroxad were applied in areas that may result in spray
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drift to such environments. Because of its environmental and health safety, as well
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as its prominent pesticide efficiency, fluxapyroxad and other SDH inhibitor fungicides
are deemed outstanding candidates for fungal disease control in the near future.
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Gas and high-performance liquid chromatography coupled to different sorts of
detectors are the most common analytical methods that are employed nowadays for
chemical residue as well as contaminant detection. These are extremely sensitive and
selective techniques, especially when coupled to mass spectrometers, and constitute
the methods of choice for large monitoring programmes for which an extraordinary
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multiresidue capacity is demanded. Nevertheless, other analytical applications, such
as toxicological crisis, industrial control departments, dissipation and transfer studies,
or just personal assessment, require straightforward, low cost, and rapid procedures
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for which the immunochemical methods provide a powerful alternative approach.
Antibody-based techniques are very selective and highly sensitive, and can be
developed in a variety of immunoassay formats. Undoubtedly, the competitive
enzyme-linked immunosorbent assay (cELISA) is the most common immunochemical
method, probably due to its large sample throughput as well as its ability to provide
quantitative results.
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In order to develop a novel cELISA procedure, three immunoreagents are needed:
the immunizing conjugate, the antibody, and the assay conjugate. The two required
conjugates generally consist of a protein to which a functionalized derivative
mimicking the target analyte, called a hapten, is linked. This synthetic molecule is
usually prepared with a spacer arm holding a functional chemical group for covalent
coupling. Rational design of different functionalized derivatives constitutes a key step
for high-affinity and specific antibody generation as well as immunoassay
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development. Haptens that are employed for the preparation of immunizing
conjugates should preferentially display the characteristic moieties and preserve the
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electronic distribution and conformation of the target compound. However, the
optimum linker tethering site is usually difficult to predict, and intricate synthetic
procedures are most commonly required in order to prepare alternative immunizing
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haptens. On the other hand, it is well established that employing heterologous
haptens, i.e. molecules that are different from the immunizing hapten, in cELISA can
improve the sensitivity of the assay and even modulate the selectivity of polyclonal
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antibody-based immunoassays.
Hapten heterologies most commonly consist of
changes in the immunodeterminant moieties of the target molecule or a different
linker composition or tethering site. These strategies have been shown to be quite
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effective, particularly in the conjugate-coated assay format.
Nevertheless, further
studies are required aiming to increase the scientific knowledge about the structure‒
immunogenicity and structure‒assay activity relationships of haptens.
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In this study, we developed for the first time high-affinity antibodies specific to
fluxapyroxad. In order to reach this goal, we investigated the influence of two
proximate linker tethering sites over the generation of antibodies and their use as
heterologous haptens. With this aim, polyclonal antibodies were raised since they are
better representatives of the immune response. Finally, alternative cELISA procedures
were developed, and their applicability to the analysis of residues of this agrochemical
in food samples was evaluated.
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MATERIALS AND METHODS
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Reagents and Instruments. Technical fluxapyroxad was generously provided by
BASF (Ludwigshafen, Germany) and Pestanal grade of fluxapyroxad and other pesticide
standards were purchased from Sigma/Aldrich (Madrid, Spain). Compounds employed
in this work only present slight safety concerns. Nevertheless, working in a well-
ventilated fume hood is advisable for synthesis work. Bovine serum albumin (BSA)
fraction V was from Roche Applied Science (Mannheim, Germany). Ovalbumin (OVA),
horseradish peroxidase (HRP), and adult bovine serum (ABS) were purchased from
Sigma/Aldrich (Madrid, Spain). Sephadex G-25 HiTrap Desalting columns (GE
Healthcare, Uppsala, Sweden) were utilized for protein–hapten conjugate purification.
Goat anti-rabbit immunoglobulin polyclonal antibody conjugated to peroxidase was
from BioRad (Madrid, Spain). Costar flat-bottom high-binding 96-well polystyrene
ELISA plates were from Corning (Corning, NY). UV–Vis spectra of haptens and
conjugates as well as ELISA absorbances were read with a PowerWave HT from BioTek
Instruments (Winooski, VT). Microwells were washed with an ELx405 microplate
washer also from BioTek Instruments.
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Preparation of Hapten FXn. The schematic representation of the synthesis of
hapten FXn is depicted in Figure 2.
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2 3
Synthesis of ethyl 1-allyl-3-(difluoromethyl)-1H-pyrazole-4-carboxylate, 2. K CO
(278 mg, 2.012 mmol, 1.5 equiv) and allyl bromide (174 µL, 2.012 mmol, 1.5 equiv)
were added to a suspension of ethyl 3-(difluoromethyl)-1H-pyrazole-4-carboxylate, 1
(254.9 mg, 1.341 mmol) in dry acetone (3 mL) under nitrogen. The resulting mixture
was stirred at room temperature for 4 h, then diluted with water and extracted with
EtOAc. The combined organic layers were washed with brine and dried over anhydrous
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MgSO
4
, filtered, and concentrated under vacuum. The residue obtained was purified
by flash chromatography performed on silica gel 60, 230–400 mesh (Merck,
Darmstadt, Germany) , using hexane/EtOAc mixtures from 9:1 to 8:2 as eluent, to give
allyl derivative 2 as an oil (216.3 mg, 70%).
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Synthesis of 1-allyl-3-(difluoromethyl)-1H-pyrazole-4-carboxylic acid, 3. A solution of
ethyl ester 2 (215.2 mg, 0.934 mmol) in a mixture of 1 M aqueous NaOH (4 mL) and
EtOH (3 mL) was stirred at room temperature for 2 h (reaction monitored by thin layer
chromatography on F254 silica gel plates, hexane/EtOAc 1:1). The reaction mixture
was diluted with water, acidified to pH 3-5 with citric acid, and extracted with EtOAc.
The combined organic extracts were washed with brine and dried over anhydrous
MgSO , filtered, and concentrated under vacuum to afford acid 3 as a white solid
4
which was used in the next step without further purification (186.6 mg, 98.7%).
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Synthesis of 1-allyl-3-(difluoromethyl)-N-(3′,4′,5′-trifluoro-[1,1′-biphenyl]-2-yl)-1H-
pyrazole-4-carboxamide, 5. A suspension of acid 3 (186.6 mg, 0.923 mmol) and PCl5
2
(207.6 mg, 0.997 mmol, 1.08 equiv) in anhydrous Et O (4.5 mL) was stirred at room
temperature under nitrogen atmosphere until complete formation of the acid chloride
intermediate (about 1h, reaction monitored by thin layer chromatography on F254
silica gel plates, hexane/EtOAc 1:3). The solvent was evaporated under vacuum and
the residue was dissolved in anhydrous THF (1 mL) and added via cannula to a solution
of anhydrous pyridine (150 μL, 1.846 mmol, 2 equiv) and 2-aminobiphenyl 4 (206.1 mg,
o
0.923 mmol, 1 equiv) in THF (1 mL) at 0 C under nitrogen. The resulting mixture was
stirred at room temperature for 1.5 h, then diluted with water, acidified to pH 3‒4
with citric acid and extracted with EtOAc. The organic extracts were washed with a 5%
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3 4
aqueous solution of NaHCO and brine, dried over anhydrous MgSO , filtered, and
concentrated under vacuum. The residue obtained was purified by chromatography,
using hexane/EtOAc mixtures from 9:1 to 6:4 as eluent, to give, in order of elution,
unreacted 2-aminobiphenyl 4 (10.1 mg, 4.9%) and amide 5 (324.7 mg, 90.7% based on
recovered starting material) as a white solid.
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Synthesis
of
6-(3-(difluoromethyl)-4-((3′,4′,5'-trifluoro-[1,1'-biphenyl]-2-
yl)carbamoyl)-1H-pyrazol-1-yl)hex-4-enoic acid, 6. But-3-enoic acid (52.8 mg, 52.2 μL,
0.615 mmol, 3 equiv) was added to a suspension of 5 (83.4 mg, 0.205 mmol), 2nd
Generation
Grubbs
Catalyst
[(1,3-bis(2,4,6-trimethylphenyl)-2-
ruthenium]
imidazolidinylidene)dichloro(phenylmethylene)(tricyclohexylphosphine)
(10.5 mg, 0.012 mmol, 0.06 equiv) and CuI (3.0 mg, 0.016 mol, 0.08 equiv) in
o
anhydrous Et
2
O (2.4 mL) under nitrogen. The resulting mixture was stirred at 60 C for
6 h under microwave irradiation (300 W). After this time, the solvent was evaporated
under vacuum and the residue obtained was purified by chromatography. Elution, first
with CH
2
Cl
2
and then with CH
2
Cl
2
-Et
2
O 9:1, gave unreacted starting material 5 (24.8
and then with CHCl /MeOH 9:1, afforded the
mg). Further elution, first with CHCl
3
3
corresponding cross-metathesis product 6 (44.8 mg, 66.8% based on recovered
starting material) as an approximately 8:2 mixture of E/Z geometrical isomers.
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Synthesis
of
6-(3-(difluoromethyl)-4-((3′,4′,5'-trifluoro-[1,1'-biphenyl]-2-
yl)carbamoyl)-1H-pyrazol-1-yl)hexanoic acid (Hapten FXn). A solution of geometric
isomers of alkene 6 (37.3 mg, 0.08014 mmol) in a mixture of 10% Pd/C (20 mg) in
EtOAc (1.9 mL) was hydrogenated under a hydrogen pressure of 3 atm for 4 h. The
black suspension was filtered through a pad of Celite with EtOAc, the filtrate was
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concentrated in vacuum, and the residue was purified by chromatography, using
CHCl
3
/MeOH 95:5 as eluent, to give hapten FXn (29.6 mg, 79%) as a white solid, m.p.
o
132.6‒133.8 C (crystals obtained from slow evaporation from CHCl
3
solution). IR
-1
(neat) νmax (cm ): 3500—2500 (br, s), 3423 (w), 3257 (m), 3127 (w), 2952 (w), 1710
1
(m), 1654 (m), 1617 (m), 1545 (s), 1045 (s), 760 (s). H-NMR (300 MHz, CDCl
3
) δ (ppm):
8.16 (1H, d, J = 8.2 Hz, H-3 BiPh), 8.04 (1H ,s, H-5 Pz), 7.86 (1H, br t, J = 3.5 Hz, NH),
7.50–7.36 (1H, m, H-4 BiPh), 7.25–7.19 (2H, m, H-5 and H-6 BiPh), 7.06–6.94 (2H, m, H-
2′ and H-6′ BiPh), 6.65 (1H, t, J = 54.2 Hz, CHF ), 4.13 (2H, d, J = 7.1 Hz, H-5), 2.39 (2H, t,
2
19
J = 7.2 Hz, H-2), 2.00-1.86 (2H, m, H-4), 1.74-1.56 (2H, m, H-3). F-NMR (282 MHz,
CDCl
) δ (ppm): −109.0 (2F, s, CHF ), −134.2 (2F, d, J = 20.6 Hz, F-3′ and F-5′ BiPh),
−162.0 (1F, t, J = 20.6 Hz, F-4′ BiPh). C-NMR (75 MHz, CD D) δ (ppm) 176.9 (CO H),
3
2
1
3
3
2
163.1 (CONH), 152.2 (ddd, J = 248.8, 10.2, 4.5 Hz, C-3′ and C-5′ BiPh), 147.0 (t, J = 24.7
Hz, C-3 Pz), 140.3 (dt, J = 250.0, 15.4 Hz, C-4′ BiPh), 137.3 (td, J = 8.5, 5.1 Hz, C-1′ BiPh),
137.0 (C-2 BiPh), 135.2 (C-1 BiPh), 133.4 (C-5 Pz), 131.4 (C-4 BiPh), 130.2 (C-5 BiPh),
128.8 (C-3 BiPh), 128.4 (C-6 BiPh), 116.7 (C-4 Pz), 114.3 (dd, J = 14.6, 6.8 Hz, C-2′ and C-
6′ BiPh), 111.2 (t, J = 235.1 Hz, CHF
2
), 53.5 (C-5), 34.1 (C-2), 30.4 (C-4), 22.9 (C-3). HRMS
+
(TOF MS ES+): m/z calcd for C H F N O [M+H] 468.1341, found 468.1340.
2
2 19 5 3 3
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Preparation of Hapten FXc. The schematic representation of the synthesis of
hapten FXc is depicted in Figure 3.
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Synthesis of 3-(dibromomethyl)-1-methyl-N-(3′,4′,5'-trifluoro-[1,1'-biphenyl]-2-yl)-
3 2 2
1H-pyrazole-4-carboxamide, 7. A solution 1 M of BBr in CH Cl (3.15 mL, 3.15 mmol, 6
equiv) was dropwise added to a solution of fluxapyroxad (200 mg, 0.525 mmol) in
o
anhydrous CH
2
Cl
2
(8 mL) at −78 C under nitrogen. The reaction mixture was allowed to
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warm slowly to room temperature and was stirred for 4 h. The mixture was then
o
cooled to 0 C, then carefully quenched with water and extracted with CH
2
Cl
2
. The
, and
combined organic extracts were washed with brine, dried over anhydrous MgSO
4
concentrated under reduced pressure to give dibromide 7 (251 mg, 95.1%) as a solid,
which was deemed sufficiently pure to be used in the next step without any further
purification.
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2
2
2
2
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03
04
Synthesis of 3-formyl-1-methyl-N-(3′,4′,5'-trifluoro-[1,1'-biphenyl]-2-yl)-1H-pyrazole-
4-carboxamide, 8. A solution of dibromide 7 (182 mg, 0.362 mmol) in anhydrous
o
pyridine (500 μL) was stirred at 100 C under nitrogen for 90 min. The resulting mixture
was cooled and poured into ice-cold water and extracted with EtOAc, the combined
organic layers were washed with water and brine and dried over anhydrous MgSO4.
Evaporation of the solvent under reduced pressure and filtration through a small plug
of silica gel, washing with CH
mg, 91%).
2 2
Cl /EtOAc 9:1, afforded nearly pure aldehyde 8 (118.5
2
2
2
2
2
2
2
2
2
05
06
07
08
09
10
11
12
13
Synthesis of (E/Z)-6-(1-methyl-4-((3′,4′,5'-trifluoro-[1,1'-biphenyl]-2-yl)carbamoyl)-
1H-pyrazol-3-yl)hex-5-enoic acid, 9. A suspension of 5-(triphenylphosphonio)pentanoic
acid bromide (144.5 mg, 0.326 mmol,
2
equiv) in anhydrous
hexamethylphosphoramide (442 μL) and THF (600 μL) was sonicated at room
temperature under nitrogen until a clear solution was obtained. Then, a 0.5 M solution
of potassium hexamethyldisilazide in toluene (1.32 mL, 0.660 mmol, 4 equiv) was
dropwise added under the same conditions to obtain a deep orange solution of the
corresponding Wittig reagent (ylide). The above mixture was cooled with stirring in an
ice-water bath, and then a solution of aldehyde 8 (58.6 mg, 0.163) in anhydrous THF
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2
2
2
2
2
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(600 μL) was added via cannula. After 1.5 h, the reaction mixture was poured into ice-
water, acidified to pH 2‒3 with 1 M HCl and extracted with EtOAc. The combined
organic layers were washed with a saturated aqueous solution of LiCl and brine, dried
over anhydrous MgSO
4
, and concentrated under reduced pressure to give an oily
/MeOH mixtures
residue (193 mg) that was purified by chromatography, using CHCl
3
from 100:0 to 95:5 as eluent, to afford alkene 9 (49.2 mg, 68%) as a thick oil. According
1
to its H-NMR spectrum, it was a ca. 3:2 mixture of E- and Z-isomers of the double
bond.
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
22
23
24
25
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31
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Synthesis of 6-(1-methyl-4-((3′,4′,5'-trifluoro-[1,1'-biphenyl]-2-yl)carbamoyl)-1H-
pyrazol-3-yl) hexanoic acid (Hapten FXc). To a solution of 9 (44.8 mg, 0.101 mmol) in
EtOAc (2 ml) was added Pd/C (10%, 23 mg) and the mixture was hydrogenated at 3
atm for 4.5 h. The catalyst was filtered through a pad of silica gel and the filter cake
was washed with EtOAc. The combined filtrates were concentrated under vacuum to
o
give hapten FXc (41.2 mg, 92.6%) as a white solid, m.p. 104.4‒105.8 C (from
–1
3
hexane/EtOAc/CHCl ). IR (neat) νmax (cm ): 2500‒3350 (br, s), 3331 (s), 3122 (w), 3073
(w), 2945 (m), 1741 (s), 1697 (s), 1670 (s), 1615 (s), 1532 (s), 1489 (s), 1425 (s), 1207
1
(s), 1038 (s), 760 (s). H-NMR (300 MHz, CDCl ) δ (ppm): 8.20 (1H, d, J = 8.2 Hz, H-3
3
BiPh), 7.50 (1H, s, H-5 Pz), 7.48-7.36 (1H, m, H-4 BiPh), 7.25‒7.12 (3H, m, H-5 and H-6
BiPh and NH), 7.12‒6.93 (2H, m, H-2′ and H-6′ BiPh), 3.83 (3H, s, NMe), 2.67 (2H, t, J =
7.5, H-6), 2.33 (2H, t, 2H, t, J = 7.5, H-2), 1.64 (4H, quint, J = 7.5, H-3 and H-5), 1.35 (2H,
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m, H-4). F-NMR (282 MHz, CDCl
BiPh), −160.9 (1F, t, J = 20.6 Hz, F-4′ BiPh). C-NMR (75 MHz, CDCl
(CO H), 161.4 (CONH), 153.0 (C-3 Pz), 151.6 (ddd, J = 252.3, 10.0, 4.1 Hz, C-3′ and C-5′
BiPh), 139.5 (dt, J = 253.7, 14.9 Hz, C-4′ BiPh), 134.5 (td, J = 7.7, 5.0 Hz, C-1′ BiPh),
3
) δ (ppm): −133.0 (2F, d, J = 20.6 Hz, F-3′ and F-5′
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) δ (ppm): 178.6
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134.7 (C-2 BiPh), 131.9 (C-5 Pz), 130.2 (C-1 BiPh), 131.1 (C-1 BiPh), 130.1 (C-6 BiPh),
129.7 (C-4 BiPh), 125.0 (C-5 BiPh), 123.2 (C-3 BiPh), 115.4 (C-4 Pz), 113.8 (dd, J = 14.6,
6.8 Hz, C-2′ and C-6′ BiPh), 39.2 (NMe), 33.9 (C-2), 28.9 (C-4), 28.2 (C-6), 27.5 (C-5),
+
24.5 (C-3). HRMS (TOF MS ES+): m/z calcd for C23
H
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F N
3
O
2
[M+H] 446.1686, found
446.1681.
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Preparation of Protein Conjugates. The synthesis of N-hydroxysuccinimidyl esters
of haptens FXn and FXc (Figures 2 and 3) was performed by reaction of the purified
hapten with N,N’-disuccinimidyl carbonate and Et N in anhydrous CH CN. Both
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haptens were covalently linked to BSA, OVA, and HRP using a 24, 8, and 5.5 molar
excess, respectively, of the corresponding purified N-hydroxysuccinimidyl ester
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according to previously established procedures. The obtained hapten-to-protein
molar ratio (MR) of the prepared conjugates was determined by MALDI-TOF mass
spectrometry.
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Antibody Generation. Procedures for animal immunization were approved by the
Ethics Committee of the University of Valencia for Animal Experimentation and
Welfare. Animal manipulation was carried out according to Spanish and European laws
and guidelines concerning the protection of animals used for scientific purposes (RD
1201/2005, Law 32/2007, and European Directive 2010/63/EU). Two white female
rabbits of around 2 kg each were subcutaneously immunized with 300 µg of BSA‒FXn
or BSA‒FXc conjugate in a 1:1 emulsion between phosphate buffer solution and
Freund’s adjuvant (complete for the first injection and incomplete for further boosts).
The immunogen was given 4 times with 21-day intervals. Whole blood was taken 10 d
after the fourth injection and it was allowed to coagulate overnight at 4 °C. The serum
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was separated by centrifugation and antibodies were precipitated twice with 1 volume
of a saturated ammonium sulfate solution. Precipitated antisera were stored at 4 °C,
and a working antibody solution was prepared by 4-fold dilution with PBS containing
1% (w/v) BSA and kept at 4 °C.
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Immunoassay Development. Immunoassays were developed using the antibody-
coated direct format and the conjugate-coated indirect format according to previously
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published procedures. Starting from a 10 µM fluxapyroxad solution in PBS, eight-
point fluxapyroxad standard curves, including a blank, were prepared by 10-fold serial
dilution in PBS. A 10 mM fluxapyroxad stock solution in anhydrous N,N-
dimethylformamide was used to prepare the first standard solution. For sample
analysis, fruit juices were purchased from local supermarkets. The competitive
reaction was carried out with samples diluted in MilliQ water and the corresponding
immunoreagent in 2×PBS containing 0.05% (v/v) Tween 20. In order to ascertain the
minimum sample dilution to obtain a reliable determination of fluxapyroxad, the
analytical parameters of the standard curves prepared in the diluted matrices were
compared to those from curves run in water. Recovery studies with fluxapyroxad
fortified fruit samples were performed in order to examine the accuracy and precision
of the developed immunoassays. Each sample was analyzed in duplicate wells and
average absorbance values were interpolated into the standard curve run in triplicate
wells in the same plate.
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Data Treatment. Microplates were read at 492 nm using 650 nm as reference
wavelength with a PowerWave HT instrument (BioTek, Winooski, VT). Experimental
values were fitted to a four-parameter logistic equation using the SigmaPlot software
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package from SPSS Inc. (Chicago, IL). Assay sensitivity was defined as the concentration
of analyte at the inflection point of the fitted curve. This value typically corresponds to
a 50% reduction (IC50) of the maximum absorbance (Amax). The limit of detection (LOD)
and the dynamic range of the assay were estimated as the concentration of analyte
providing a decrease of Amax of 10% and between 20% and 90%, respectively. Cross-
reactivity was determined as the percentage of the quotient between the IC50 value for
fluxapyroxad and the IC50 value for the evaluated compound.
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Sample Preparation. Peach, apple, and grape juices were obtained from local
supermarkets. In order to obtain contaminated samples, grapevines were treated with
a commercial formulation kindly provided by BASF Crop Protection (Sercadis,
fluxapyroxad 300 g/L) at the dose recommended by the manufacturer using a manual
sprayer. Three days after the treatment, grape samples (500 g) were harvested, and
upon arrival at the laboratory berries were separated from the stems, homogenized
using a T-25 Ultra-Turrax blender (IKA, Staufen, Germany), and stored in polypropylene
tubes at −20 °C until analysis. The QuEChERS method was employed for extraction of
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fungicide residues from samples. Briefly, homogenized grapes (5 g) were introduced
into 50 mL polypropylene centrifuge tubes containing 0.5 g of sodium acetate and 2.0
g of anhydrous magnesium sulfate. Samples were then extracted by vortexing for 1
min with 5 mL of 1% (v/v) acetic acid in acetonitrile containing 500 µg/L of
triphenylphosphate as internal standard, and centrifuged for 5 min at 2200×g. The
extracts (1 mL) were then cleaned-up with 50 mg of PSA (primary‒secondary amine)
and 150 mg of anhydrous magnesium sulfate, vortexed for 1 min, and centrifuged 5
min at 6700×g. Purified extracts were then filtered through a 0.22 µm Teflon filter and
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analyzed by the optimized cELISA and by ultra-performance liquid chromatography
coupled to a triple quadrupole tandem mass spectrometer (UPLC–MS/MS).
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Chromatographic analysis. Determinations were carried with an Acquity UPLC
system (Waters, Milford, MA) equipped with a 50 mm × 2.1 mm i.d., 1.7 µm, Bridged
Ethylene Hybrid C18 column. The injection volume was 5 µL and the mobile phase
consisted of 0.5% (v/v) formic acid in water (A) and acetonitrile (B). The elution
gradient started at 50% of B at a flow-rate of 400 µL/min, and then B was linearly
increased to 95% in 4 min. The obtained retention times, using the aforementioned
conditions, were 1.36 and 2.36 min for fluxapyroxad and triphenylphosphine as
internal standard, respectively. Tandem mass acquisitions were performed in a Waters
Acquity triple quadrupole mass spectrometry detector, equipped with a Z-spray
electrospray ionization source, with 3.5 kV capillary voltage, at 120 °C as source
temperature, and 300 °C as desolvation temperature. The employed parameters were:
ESI+, parent ion m/z 382.0, daughter ions m/z 314.3 and 342.3, 30 eV collision energy,
and 20 V cone energy for fluxapyroxad; and ESI+, parent ion m/z 328.3, daughter ions
m/z 77.0 and 152.4, 30 eV collision energy, and 25 V cone energy for
triphenylphosphine.
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RESULTS AND DISCUSSION
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Hapten Synthesis. Two functionalized haptens mimicking fluxapyroxad were
designed, namely haptens FXn and FXc (Figures 2 and 3). Both haptens incorporate a
spacer arm suitable for protein conjugation, namely a carboxylated hydrocarbon linear
chain, in complementary positions of the pyrazole ring. The spacer arm in hapten FXn
was a substituent of the methyl group at N-1 which would produce an insignificant
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effect on the electronic distribution and conformational mobility of the fluoxapyroxad
framework. In the case of hapten FXc, the incorporation of the alkyl chain as a C-3
substituent of the difluoromethyl group would entail a greater change, both in the
electronic distribution of the pyrazole ring and in the conformational disposition of the
amide group, due to the weak electron-donor effect of the alkyl chain compared to the
withdrawing electron effect of the difluoromethyl group and the greater steric
hindrance introduced by the highly flexible linear hydrocarbon spacer arm.
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The preparation of hapten FXn is outlined in Figure 2. The synthesis began with the
incorporation of an allyl group at the N-1 position of ethyl 1H-pyrazole-4-carboxylate 1,
which was used as both a blocking group of this position during the construction of the
fluxapyroxad framework and as a pivotal moiety for the subsequent elaboration of the
hydrocarbon chain constituting the spacer arm. Thus, alkylation of 1 with allyl bromide
under basic conditions led to allyl ether 2, whose ethyl ester moiety was hydrolyzed
under conventional basic conditions to afford pyrazole carboxylic acid 3. Activation of
the carboxylate group to the acid chloride, by treatment with phosphorus
pentachloride in ether at room temperature, followed by reaction with biphenyl amine
4 in the presence of pyridine, to quench the acid (HCl) formed, led to amide 5, thus
completing the part of the hapten skeleton common with the parent analyte.
Elaboration of the C5 carboxylated chain at N-1 from 5 was carried out via a
microwave irradiation promoted cross-metathesis reaction of the allyl ether moiety
with but-3-enoic acid catalyzed by Grubbs' second generation catalyst and copper
iodide as co-catalyst. The cross-metathesis reaction afforded alkene acid 6 as a ca. 8:2
mixture of E/Z geometrical isomers of the double bond that was hydrogenated under
heterogeneous catalyzed conditions to give the desired hapten. The synthesis of
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hapten FXn was thus completed from pyrazole 1 in six synthetic steps and 31% overall
yield.
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On the other hand, the synthesis of hapten FXc was initiated from fluxapyroxad and
involved the initial transformation of the difluoromethyl group into a dibromomethyl
group (Figure 3), which underwent hydrolysis to a formyl group by heating in pyridine
and subsequent aqueous workup to afford aldehyde 8. The elaboration of the
carboxylated spacer arm was carried out, in this case, through a Wittig olefination
reaction between the aldehyde carbonyl group of 8 and the ylide derived from (4-
carboxybutyl)triphenylphosphonium bromide and potassium hexamethyldisilazane,
which afforded the olefinic acid 9 as a ca. 3:2 mixture of trans and cis isomers,
followed by catalytic hydrogenation of the generated double bond. The synthesis of
hapten FXc was thus achieved in four steps with an excellent 63% overall yield from
fluxapyroxad.
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Immunoreagent Preparation. The same coupling procedure, the active ester
method, was applied for preparation of both the immunizing and the assay conjugates.
Previously, the carboxyl functional group of each hapten was activated under smooth
conditions with N,N′-disuccinimidyl carbonate, which allowed the easy isolation and
further chromatographic purification of the corresponding active esters, FXn-NHS and
FXc-NHS esters (Figures 2 and 3). Freshly prepared and purified succinimidyl esters of
haptens were used for covalently coupling to BSA, OVA, and HRP. Since the pure active
ester was employed, no succinimide and carbodiimide were present during the
coupling reaction; thus, no secondary and cross-coupling reactions could occur and
higher coupling yields could be accomplished, so lower amounts of hapten were
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required. The obtained hapten-to-protein molar ratios (MR), determined by MALDI-
TOF mass spectrometry, for BSA, OVA, and HRP conjugates were 18.9, 5.3, and 1.2 and
15.2, 5.4 and 1.3 for haptens FXn and FXc, respectively (Figure 4). These values are
within the optimum range of MR values for immunizing and assay conjugates.
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Four rabbits were immunized; two with conjugate BSA‒FXn and two with conjugate
BSA‒FXc. Thus, two polyclonal antibodies, named FXn#1 and FXn#2, were obtained
from the conjugate of hapten FXn, and two, named FXc#1 and FXc#2, from the
conjugate of hapten FXc.
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Antibody Characterization. Specificity of the antibodies was assessed by indirect
cELISA using the homologous coating conjugate. All of the generated polyclonals were
highly specific of fluxapyroxad. Inhibition was negligible even with a concentration of
10 µM of other SDH-inhibitor fungicides (penthiopyrad and fluopyram) as well as other
widely used pesticides which may be present together with fluxapyroxad in real food
samples (azoxystrobin, picoxystrobin, pyraclostrobin, kresoxim-methyl, fenhexamid,
fluopicolide, pyrimethanil, and cyprodinil). Only a slight binding was observed with
boscalid. Interestingly, the cross-reactivity was 10 times lower with antibodies
obtained from hapten FXn than with FXc-type antibodies, indicating the relevance of
the linker tethering site for antibody specificity.
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Antibody affinity was evaluated by checkerboard cELISA using homologous (same
hapten as that of the immunizing conjugate) and heterologous (the hapten was
different from the immunizing conjugate) conjugates in two different assay
procedures: the antibody-coated direct format and the conjugate-coated indirect
format (Table 1). According to homologous assays, little difference was observed
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between the titers (antibody dilution affording an Amax value around 1.0 in the absence
of analyte in solution) of FXn- and FXc-type antibodies, and high-affinity antibodies to
fluxapyroxad were obtained from both of the immunizing haptens, with IC50 values
generally in the low nanomolar range. The only exception was antibody FXc#1 which
showed a high IC50 value in the indirect homologous assay. Therefore, the pyrazole ring
seems to be a suitable linker tethering site in the fluxapyroxad molecule for high-
affinity antibody generation. The lowest IC50 value using homologous conjugates was
obtained with antibody FXn#2 in both assay formats. This result, together with the
higher specificity of FXn-type antibodies, indicated a slight superiority of hapten FXn to
elicit the best immune response. This finding could be explained by the minor changes
introduced by the spacer arm in hapten FXn compared to the non-negligible changes in
the electronic and conformational properties of the pyrazole ring produced by the
linker in hapten FXc as discussed above.
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It is well established that heterologous conjugates can help to improve
immunoassay sensitivity. However, if too drastic heterologies are used, binding of the
antibody to the assay conjugate could be insufficient, particularly in the antibody-
coated format. The two haptens (FXn and FXc) prepared in this study held the spacer
arm in proximal positions, so a similar orientation of the distal moieties was expected.
As envisioned, all of the antibodies recognized the corresponding heterologous
conjugate, including the enzyme tracer in the direct assay format, with the same or
similar titers (Table 1). The slope of the fitted inhibition curve was generally between
0.8 and 1.0, and the background signal was always below 0.1. Mostly, assay sensitivity
could be enhanced by using the heterologous conjugates; the IC50 values were
generally reduced around 50% or more if compared to the homologous assay. The
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differences in the electronic and structural properties between both haptens did not
seem to hinder antibody binding. These results are in accordance with the
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Landsteiner’s principle stating that the specificity of antibodies is mainly directed
towards the distal moieties of small organic molecules.
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The most sensitive immunoassays were found with antibody FXn#2 in combination
with the heterologous conjugate, both in the direct and indirect assay formats. The
main analytical parameters of the standard curve of the best immunoassay in each
format are listed in Figure 5. Both standard curves showed Amax values over 1.0 and
slope values close to 1.0, which constitute excellent parameters for immunoassay
application. The IC50 for fluxapyroxad of the direct and indirect assays were 0.40 nM
(0.15 µg/L) and 1.51 nM (0.58 µg/L), respectively. The calculated LOD values were 0.05
nM (0.019 µg/L) and 0.12 nM (0.046 µg/L) for the direct and indirect assay,
respectively. Therefore, in this particular case, the sensitivity of the direct assay was
slightly better than that of the indirect assay, probably due to the antibody
characteristics. Generally, when polyclonal antibodies are used, heterologous
conjugates enhance assay sensitivity of both cELISA formats, and similar sensitivities
are usually attained with either format, as previously reported for other small chemical
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compounds.
Finally, the theoretical dynamic working range was established
between 0.09 nM (0.034 µg/L) and 4.30 nM (1.640 µg/L) for the direct assay, and
between 0.30 nM (0.114 µg/L) and 17.89 nM (6.820 µg/L) for the indirect format. Due
to their similar sensitivities, the two optimized immunoassays were further evaluated
for food sample analysis.
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Sample Analysis. Matrix effects of peach, apple, and grape juices were determined
by running standard curves of fluxapyroxad in juices diluted in water (from 1/5 to
1/500). Very low interferences were seen with peach and apple juices in the direct
assay, whereas grape juice seemed to induce slight matrix effects (Figure S1). On the
other hand, similar results were found with the indirect assay, where the grape juice
showed the strongest interference (Figure S2). In all cases, a 1/25- or a 1/50-fold
dilution seemed to minimize the observed matrix effects.
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Next, peach, apple, and grape juices were fortified with fluxapyroxad and analyzed
by the developed immunoassays. Table 2 lists the recovery rates and the coefficients
of variation found by the direct and indirect cELISA. For the former assay, precise and
accurate results were obtained for all of the juices in the range from 5 to 500 µg/L,
with just a 1/25-fold sample dilution in water. Coefficients of variation higher than 20%
were only seen for the lowest and the highest assayed concentration in apple and
grape juice, respectively. For all three types of samples, the limit of quantitation (LOQ)
of the direct immunoassay could be set at 5 µg/L. Regarding the indirect assay, a 1/50-
fold sample dilution in water was required in order to obtain accurate and precise
results (Table 2). Somewhat unprecise results were found with apple juice samples at
the lowest assayed concentrations. On the contrary, good recovery rates and
acceptable coefficients of variation were found for peach and grape juices spiked with
fluxapyroxad at concentrations between 30 and 3000 µg/L. Therefore, the LOQ of the
indirect immunoassay could be established at 300 µg/L for apple juice and at 30 µg/L
for peach and grape juices. Consequently, the LOQs displayed by the developed direct
and the indirect cELISAs were much lower than the maximum residue limits (MRL)
established for peach, apple, and grape samples by the European Commission (1500,
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900, and 3000 µg/L, respectively) and by the US Department of Agriculture (3000,
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800, and 2000 µg/L, respectively). Nevertheless, in terms of sensitivity and
robustness, the direct assay seemed to be the best choice for the immunoanalysis of
fluxapyroxad residues in foodstuffs.
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Due to the lack of juice samples with incurred residues and in order to provide
compelling evidences of the suitability of the direct cELISA for the determination of
fluxapyroxad residues under real conditions, a limited number of grapevines were
treated with a commercial formulation of the fungicide during the 2015/2016 season
near the harvest period. Grape samples were harvested three days after the treatment
with fluxapyroxad and extracted using the QuEChERS method. Extracts were directly
analyzed by UPLC–MS/MS and by cELISA after 500-fold dilution in MilliQ water. Linear
regression analysis indicated an acceptable correlation between the developed direct
immunoassay and the reference chromatographic method (y = 1.09x + 15.12;
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r =0.963), with only a slight deviation between both sets of measurements (Figure 6).
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In summary, two haptens mimicking the molecule of fluxapyroxad were prepared
with the linker arm located at the same ring. Moreover, bioconjugates to different
proteins were obtained with adequate yields by the active ester method using the
purified activated hapten, and antibodies to fluxapyroxad were generated for the first
time. A slightly higher specificity was observed for those binders that were obtained
from the hapten with the spacer arm at the N-1 position of the pyrazole ring. High-
affinity to fluxapyroxad was observed by cELISA, with IC50 values in the low nanomolar
range. Two immunoassays were developed in different formats with antibody FXn#2
and using a heterologous conjugate. In the analysis of fruit juices, the direct assay
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outperformed the indirect assay in terms of sensitivity and robustness, so it was
applied to the quantitation of grape samples with incurred residues of fluxapyroxad,
with satisfactory results.
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ASSOCIATED CONTENT
Supporting Information
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General experimental procedures and techniques, preparation of 3',4',5'-trifluoro-
[1,1'-biphenyl]-2-amine (4), characterization data of intermediates of the synthesis of
hapten FXn, characterization data of intermediates of the synthesis of hapten FXc,
hapten activation and characterization of active esters, preparation of bioconjugates,
MALDI mass spectrometry analysis of immunizing and assay bioconjugates,
competitive ELISA procedures, antibody specificity (Table S1), juice matrix effects over
the direct immunoassay (Figure S1), juice matrix effects over the indirect immunoassay
1
(Figure S2), H-NMR spectra of haptens. This material is available free of charge via de
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
Tel: +34-963900022; Fax: +34-963636301; E-mail: aabad@iata.csic.es
ORCID iD
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Josep V. Mercader: 0000-0002-1838-2647
Antonio Abad-Somovilla: 0000-0002-5599-3682
Consuelo Agulló: 0000-0001-6056-5087
Antonio Abad-Fuentes: 0000-0001-5672-1438
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Funding
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This work was supported by the Spanish Ministerio de Ciencia e Innovación (AGL2012-
39965-C02 and AGL2015-64488-C2) and cofinanced by European Regional
Development Funds.
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ACKNOWLEDGEMENTS
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Conjugate analysis was carried out at the Proteomics Unit – a member of ISCIII
ProteoRedProteomics Platform – and animal manipulation was carried out at the
Animal Production Section, both belonging to the SCSIE of the University of Valencia.
The collaboration of Javier Fernández Abellán in providing grapevine plots for fungicide
treatments is greatly appreciated. Limited amounts of the described immunoreagents
are
available
upon
request
for
evaluation.
http://www.haptens-
antibodies.com/en/descripcion-de-la-tecnologia/.
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