Development of a monoclonal antibody-based ELISA for the detection of the novel insecticide cyantraniliprole

Article abstract of DOI:10.1039/c5ra01127b

Cyantraniliprole is the newest anthranilic diamide insecticide acting on ryanodine receptors with higher efficacy, and broader spectrum than chlorantraniliprole in pest control. To study the effects of its residues on the environment and food samples, an

Full text of DOI:10.1039/c5ra01127b

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Development of a monoclonal antibody-based
ELISA for the detection of the novel insecticide
cyantraniliprole†
Cite this: RSC Adv., 2015, 5, 35874
a
b
ab
a
a
a
Rui Zhang,‡ Kechang Liu,‡ Yongliang Cui,‡ Wei Zhang, Lishan He, Suoqin Guo,
a
c
b
a
Yuanyuan Chen, Qing X. Li, Shangzhong Liu* and Baomin Wang*
Cyantraniliprole is the newest anthranilic diamide insecticide acting on ryanodine receptors with higher
efficacy, and broader spectrum than chlorantraniliprole in pest control. To study the effects of its
residues on the environment and food samples, an enzyme-linked immunosorbent assay (ELISA) has
been developed. The hapten was synthesized and conjugated with bovine serum albumin (BSA) and
ovalbumin (OVA) as the immunogen and coating antigen, respectively. A sensitive and specific
2 2
monoclonal antibody (mAb) was generated and was designated as mAb3B . mAb3B was used to
develop an indirect competitive ELISA (icELISA). The concentration of cyantraniliprole producing 50%
ꢀ
1
ꢀ1
inhibition (IC50) was 1.57 mg L and the effective range of icELISA was 0.43–6.15 mg L . The icELISA
showed low cross-reactivity with chlorantraniliprole, and very low or no cross-reactivity with the other
cyantraniliprole analogues. Average recoveries from pakchoi (Brassica rapa) and tap water samples were
Received 20th January 2015
Accepted 13th April 2015
9
3.7–101.0% and 94.4–100.6%, respectively. Meanwhile, to confirm the results of icELISA, high-
performance liquid chromatography was applied for the determination of cyantraniliprole residues in
pakchoi samples. The comparable results suggest that the ELISA is suitable for rapid detection of
cyantraniliprole residues in environmental and agricultural samples.
DOI: 10.1039/c5ra01127b
www.rsc.org/advances
3
–5
in chlorantraniliprole.
Cyantraniliprole,§ as the newest
1
. Introduction
anthranilic diamide insecticide, also can activate the ryano-
The ongoing discovery and development of insecticides dine receptors of insect pests, and cause calcium ions to pass
with a novel mode of action is vital for ensuring the sustain- through the muscle cells rapidly, resulting in feeding cessa-
1
,2
ability of crop protection strategies. Compounds having a tion, muscle paralysis, lethargy and regurgitation like chlor-
6
,7
diamide skeleton structure like insecticides chloran- antraniliprole.
It could be against many important
traniliprole and ubendiamide could bind to the ryanodine agricultural pests, such as Coleoptera, Diptera, Isoptera, and
receptors of insect pests and became a new class of insecti- Hemiptera with low mammalian toxicity. Thus, cyan-
cide. Chlorantaniliprole has been extensively employed to traniliprole can be applied on more crops to control pests in
control lepidoptera pests on crops due to its excellent efficacy, comparison with chlorantraniliprole and had the potential to
6
low toxicity and unique action mode. However, the narrow reduce transmission of tomato spotted wilt virus.
control spectrum and no systemic character of chlor-
Cyantraniliprole can be degraded easily and the nal
8
antaniliprole limit its application at some extent. To solve residual levels in pakchoi and soil were low. Even though it is
these two issues, DuPont discovered cyantraniliprole through environmentally friendly, some studies reported that it still has
9,10
replacing chlorine atom with cyano group on the benzene ring some harmful effects, especially in agricultural system.
Furthermore, possible improper use during a harvest period
could result in a level of residues being harmful to humans.
Misuses could lead the rapid development of pest resistance.
The levels of cyantraniliprole residues in food or the environ-
a
College of Agriculture and Biotechnology, China Agricultural University, Beijing
1
00193, China. E-mail: wbaomin@263.net; Fax: +86-10-62732567; Tel: +86-10-
2731305
6
b
College of Science, China Agricultural University, Beijing 100193, China. E-mail: ment are, therefore, strictly regulated by regulatory authorities.
shangzho@cau.edu.cn; Tel: +86-10-62731070
The Pest Management Regulatory Agency (PMRA) prescribed
c
ꢀ1
Department of Molecular Biosciences and Bioengineering, University of Hawaii at
0
.01 mg g as the maximum concentration of cyantraniliprole
Manoa, Honolulu, Hawaii 96822, USA
ꢀ1
residues in meat, egg and dairy products, and 30 mg g on leafy
†
Electronic supplementary information (ESI) available:
H C
d , d and HRMS
spectrums of cyantraniliprole hapten (F ). For review only. See DOI:
6
1
0.1039/c5ra01127b
§ 3-Bromo-1-(3-chloro-2-pyridinyl)-N-[4-cyano-2-methyl-6-[(methylamino) carbonyl]-
phenyl]-1H-pyrazole-5-carboxamide.
‡
Rui Zhang, Kechang Liu, Yongliang Cui contributed equally to this work.
35874 | RSC Adv., 2015, 5, 35874–35881
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
11
brassica vegetables. Such low maximum residue limits bring Acetonitrile in chromatography grade was purchased from Fisher
challenges to the inspection and monitoring. Therefore, it is Scientic (New Jersey, USA). All other reagents were obtained
indispensable to establish a quick determination method to from Beijing Chemical Reagents Co. (Beijing, China).
monitor its residues and degradation rate in food and the
environment. Analytical methods based on high-performance
liquid chromatography-tandem mass spectrometry (HPLC-MS/
MS) and ultra-performance liquid chromatography-tandem
mass spectrometry (UPLC-MS/MS) methods have been estab-
2
.2. Apparatus
Cell culture plates and 96-well polystyrene microplates were
purchased from Costar (Corning, NY). The electric heating
constant-temperature incubator was purchased from Shanghai
Zhicheng Analytical Instrument Manufacturing Co. Ltd.
7,8
lished to measure the residue level, by which the limit of
ꢀ1
quantitation (LOQ) was 0.01 mg g in four matrix groups of oily,
(Shanghai, China). The automated plate washer (Wellwash 4
7
watery, acidic, and dry crop. The chromatographic and spec-
MK2), microplate reader (Multiskan MK3) and direct heat CO
incubator (Thermo, 311) were purchased from Thermo (Vantaa,
2
trophotometric methods show a good sensitivity and accuracy,
while they are expensive, time-consuming, and usually need
Finland). The Agilent 1200 HPLC system and 5 mm ꢁ 4.6 mm ꢁ
12,13
extensive sample preparation.
Therefore, it is of great
150 mm Betasil C column (Agilent, USA) were used for pesti-
18
signicance to propose an efficient and cost-effective method
to inspect cyantraniliprole residues in food and the environ-
ment. Immunoassays, as an alternative and complementary
cide analysis. The 0.22 mm syringe lters were purchased from
Pall (Ann Arbor, MI).
15–17
method for the analysis of agrochemicals,
are based on the
2.3. Buffers and solutions
interaction of an analyte with an antibody that recognizes the
14
analyte with high affinity and specicity. Enzyme-linked The buffers and solutions included coating buffer (0.05 M
immunosorbent assay (ELISA) is an immunoassay and has carbonate buffer, pH 9.6), phosphate-buffered saline (PBS)
been extensively applied in the detection of environmental (0.1 M phosphate buffer containing 0.9% sodium chloride, pH
12,18–23
contaminants.
Compared to conventional instrumental 7.5), PBS with 0.1% (v/v) Tween-20 (PBST), PBST containing
methods, immunoassay is a rapid and sensitive method which 0.5% (w/v) gelatin (PBSTG), citrate-phosphate buffer (0.01 M
needs small quantities of test materials and simple pretreat- citric acid and 0.03 M monosodium phosphate, pH 5.5),
ments. These features convert ELISA into very powerful tools for substrate solution (4 mL of 30% H O added to 10 mL of citrate-
2
2
ꢀ1
such pesticides that are difficult or costly to determine due to phosphate buffer containing 2 mg mL OPD), a stop solution
their physicochemical characteristics and for large monitoring (2.0 M sulfuric acid) and extracting solution (0.1 g butylated
14
programs.
hydroxyltoluene added to the mixture of 100 mL distillated
To our knowledge, there is no report of monoclonal antibody water and 400 mL absolute methanol). All reagents and solvents
(mAb)-based ELISA to monitor residual cyantraniliprole in the were analytical-grade. Deionized water used for making buffers
environment and food samples. In the present study, a cyan- and solutions was collected from a Millipore Water Purication
traniliprole hapten was synthesized and a sensitive and selec- System (Millipore Co., Billerica, MA).
tive indirect competitive ELISA was developed for monitoring
cyantraniliprole degradation and residues.
2.4. Myeloma cell line and medium
The HAT-sensitive Balb/c mouse myeloma cell line SP2/0-Ag14
obtained from the China Institute of Veterinary Drug Control
2
. Materials and methods
(Beijing, China) was used in fusion experiments. DMEM con-
2.1. Materials
taining 10–20% (v/v) FBS was supplemented with 0.2 M gluta-
Cyantraniliprole standard (99.0% purity) and cyantraniliprole
analogues were provided by the College of Science, China Agri-
cultural University, Beijing, China. Formulation of cyan-
traniliprole (suspension concentrate SC, 2.5%) was purchased
from Shanghai DuPont (China). Reagents purchased from Sigma-
Aldrich (St. Louis, MO) included 1-(3-dimethylaminepropyl)-3-
ꢀ
1
ꢀ1
mine, 50 000 U L penicillin, and 50 mg L streptomycin,
which was used for cultivating the myeloma and hybridoma
cells.
2.5. Hapten synthesis
24–28
ethylcarbodiimide (EDC), succinic anhydride, 4-dimethylamino- Hapten was synthesized
pyridine (DMAP), polyethylene glycol 2000, dimethyl sulfoxide illustrated in Fig. 1.
according to the synthetic route
(DMSO), hypoxanthine, aminopterin, and thymidine (HAT),
Synthesis of 2-amino-5-iodine-3-methyl benzoic acid (F
2
). 2-
penicillin, streptomycin, L-glutamine, complete and incomplete Amino-3-methyl benzoic acid (F
1
, 200 g, 1.32 mol) was dissolved
Freund's adjuvant, BSA, OVA, o-phenylenediamine (OPD), poly- in 700 mL of DMF in a 2 L ask equipped with a mechanical
ꢂ
oxyethylene sorbitan monolaurate (Tween-20), N-hydroxy- stirring, and then the solution was heated to 68 C. N-Iodo-
succinimide (NHS), N,N-dimethylformamide (DMF), and N- succinimide (NIS, 300 g, 1.33 mol) were slowly added into the
ꢂ
iodosuccinimide (NIS). Goat anti-mouse IgG conjugated with ask, and then stirred at 75 C for 3 h. The reaction mixture was
horseradish peroxidase (IgG–HRP) was purchased from Jackson slowly poured into 1 L of ice water and was stirred for 3 h. F as a
2
Immuno Research Laboratories (West Grove, PA). Cell culture gray solid (354 g, 72%) was obtained aer ltration and drying.
medium (Dulbecco's modied Eagle's medium) and fetal bovine
serum (FBS) were obtained from Gibco BRL (PaisLey, Scotland). Hz, Ar-H), 7.87 (1H, d, J ¼ 2.2 Hz, Ar-H).
d
H
(300 MHz, DMSO): 2.08 (3H, s, CH
3
), 7.44 (1H, t, J ¼ 1.1
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 35874–35881 | 35875

View Article Online
RSC Advances
Paper
ꢂ
ꢂ
Fig. 1 Route of cyantraniliprole hapten synthesis. Reagents and conditions (a) NIS, DMF, 68–75 C, 3 h; (b) CuCN, DMF, 145 C, 6 h; (c) tri-
ꢂ
ꢂ
2 2 3
phosgene, 1,4-dioxane, 120 C, 4 h; (d) pyridine, 115 C, 40 min; (e) NH (CH ) COOH, DMF, rt, 12 h.
Synthesis of 2-amino-5-cyano-3-methyl benzoic acid (F
3
). F
2
aminobutyric acid (34 mg, 0.33 mmol), and sodium hydroxide
(
138.5 g, 0.5 mol) and cuprous cyanide (58.2 g, 0.65 mol) were (24.6 mg, 0.61 mmol) were added in 7 mL of DMF. The mixture
added into 1 L of DMF in a 2 L ask, and the mixture was heated was stirred at room temperature for 12 h. The reaction mixture
ꢂ
to at 145 C and maintained for 6 h in an oil bath. Aer the was poured into 15 mL of water, acidied to pH 3 with 2 M HCl,
reaction completed, most of the solvent was removed through and then extracted with ethyl acetate (5 mL ꢁ 3). The combined
reduced pressure distillation, and then the residue was treated organic layers were washed with water (5 mL ꢁ 3), dried with
with 1 L of water and 50 mL of ethylenediamine. The mixture anhydrous sodium sulfate, ltered and concentrated. The
was ltered, and the ltrate was acidied to pH 5 with 2 M HCl residue was recrystallized with ethyl acetate and petroleum
and stirred overnight. F
obtained by ltration and drying.
(300 MHz, DMSO): 2.13 (3H, s, CH
.75 (1H, m, Ar-H), 7.97 (1H, t, J ¼ 1.06 Hz, Ar-H), 13.10 (1H, br, 2.28 (5H, m, CH
COOH).
(1H, s, Py-H), 7.58–7.62 (1H, m, CONHCH
3
as a gray solid (78.4 g, 89%) was ether to afford the cyantraniliprole hapten (F
(129 mg, 79%).
(300 MHz, DMSO): 1.64–1.68 (2H, m, CH
and CH ), 3.12–3.18 (2H, m, NHCH
), 7.78 (1H, d, J ¼ 1.8
6
) as a white solid
d
H
3
), 7.34 (2H, br, NH
2
),
d
H
2
CH
2
CH
2
), 2.21–
2
), 7.41
7
2
3
2
Synthesis of 8-methyl-2,4-dioxo-2,4-dihydro-1H-benzo[d][1,3]- Hz, Ar-H), 7.88 (1H, d, J ¼ 1.8 Hz, Ar-H), 8.14–8.17 (1H, m, Py-H),
oxazine-6-carbonitrile (F ). F (38 g, 0.22 mol) was dissolved in 8.42–8.46 (1H, m, Py-H), 8.48–8.50 (1H, m, Py-H), 10.52 (1H, br,
4
3
ꢂ
4
00 mL of 1,4-dioxane in a 1 L ask, and heated to 102 C with s, NHCO), 12.03 (1H, br, s, COOH).
an oil bath. A solution of triphosgene (22.7 g, 0.075 mol) in
(75 MHz, DMSO): 17.82, 24.33, 31.27, 38.75, 109.39,
0 mL of dioxane was slowly added into the ask, and the 111.01, 118.20, 126.73, 126.96, 127.98, 129.80, 135.02, 135.30,
d
C
5
ꢂ
mixture was kept at 102 C for 4 h aer addition. When reaction 137.32, 137.88, 139.21, 139.36, 147.22, 148.45, 155.44, 165.44,
was completed, the mixture was cooled down to room temper- 174.35.
HRMS (ESI): calc. C22
found 543.1047.
H
17BrClN
6
O
4
[M ꢀ H]ꢀ for 543.0189,
ature. A white solid F (37.8 g, 85%) was obtained aer ltration
and drying.
4
d
(300 MHz, DMSO): 2.35 (3H, s, CH ), 8.00 (1H, t, J ¼ 0.91
H
3
Hz, Ar-H), 8.21 (1H, d, J ¼ 1.89 Hz, Ar-H), 11.46 (1H, s, NH).
2.6. Preparation of cyantraniliprole–protein conjugates
Synthesis of 2-(3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazol-
The hapten was conjugated with BSA and OVA according to the
carbodiimide method to produce immunogen (cyan-
traniliprole–BSA) and coating antigen (cyantraniliprole–OVA).
5
-yl)-8-methyl-4-oxo-4H-benzo[d][1,3]oxazine-6-carbonitrile (F
A mixture of 3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole-5-
carbonyl chloride (0.64 g, 0.02 mol) and F (2.05 g, 0.1 mol)
5
).
13
4
The mixture was dialyzed against 3 L of PBS for 3 days with three
was dissolved in 20 mL of anhydrous pyridine in a 100 mL ask,
then heated to 115 C slowly with an oil bath and kept for
4
temperature, then was acidied to pH 5 with 2 M HCl, and
collected precipitate through ltration. The obtained precipi-
tate was recrystallized in mixed ethyl acetate and petroleum
ꢂ
changes per day, then lyophilized and stored at ꢀ20 C. The
ꢂ
structure of hapten conjugated to proteins was spectrophoto-
0 min. The reaction mixture was cooled down to room
metrically conrmed.
2
.7. Immunization protocol, monoclonal antibody
production, purication
), 7.31 (1H, s, Ar-H), Animal experiments were conducted under principles in good
.75–7.97 (3H, m, Ar-H), 8.3–8.5 (2H, m, Ar-H).
laboratory animal care, and approved by ethical committee for
ether (1 : 3, v/v) to get a white solid as F
5
(7.7 g, 87%).
d
H
(300 MHz, CDCl ): 1.86 (3H, s, CH
3
3
7
Synthesis of (2-(3-bromo-1-(3-chloropyridin-2-yl)-1H-pyrazole- Laboratory Animals Care and Use of China Agricultural
-carboxamido)-5-cyano-3-methylbenzamido) butanoic acid University. Six female Balb/c mice, 7 weeks old, were immu-
5
(
cyantraniliprole hapten).
F
5
(132 mg, 0.3 mmol), 4- nized with the immunogen cyantraniliprole–BSA. The protocols
35876 | RSC Adv., 2015, 5, 35874–35881
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
of immunization, fusion, antibody production, purication purchased from different local supermarkets. All collected
1
2
ꢂ
were as previously described.
samples were stored at ꢀ40 C until analysis.
mAbs were screened and identied as following:
Three days aer the last injection of a mixture of immu-
nogen and adjuvant, the mouse with the highest titer and best
specicity was boosted intraperitoneally with 0.1 mg cyan-
traniliprole–BSA conjugate in 0.1 mL PBS and was used for the
fusion. The spleen cells collected from the mouse were fused
with the SP2/0 (obtained from China Institute of Veterinary
Drug Control, Beijing, China) cell line using PEG-2000 at a ratio
of 10 : 1 of spleen to myeloma cells. The plates were incubated
2
.11. Preparation and extraction of samples
Chopped and homogenized pakchoi leaves (1 g) were accurately
weighed into a 10 mL polytetrauoroethylene centrifuge tube,
followed by addition of 8 mL extracting solution. The mixture
ꢂ
was sit at 4 C overnight, and was centrifuged for 10 min at 8000
rpm. The supernatant was dried by a gentle stream of nitrogen
gas to dryness. The residues were dissolved in PBSTG, and were
diluted 200 or 500 folds for icELISA. Each analysis was per-
formed in triplicate.
ꢂ
at 37 C in a CO incubator (5% CO in air). Selective growth of
2
2
the hybrid cells in the DMEM supplemented with HAT. Seven
days aer fusion, the supernatant was tested by icELISA. Posi-
tive hybridomas were cloned by limiting dilution and clones
were further selected by icELISA. The clone, designated as 3B
having a high antibody titer and good sensitivity in the culture
2
,
2.12. Sample fortication for recovery tests
supernatant was expanded in mice for production of MAb in One gram of pakchoi leaves was cut into pieces and then spiked
ascites. The 3B hybridoma cell line was archived in the Engi- with cyantraniliprole at concentrations of 0, 250, 500, 1000,
2
ꢀ1
neering Research Center of Plant Growth Regulator, China 2000, and 4000 ng g . Tap water was spiked at concentrations
ꢀ
1
Agricultural University.
of 0, 1.25, 2.5, 5, 8, 10 mg L cyantraniliprole in spiked samples
were analyzed with the established icELISA.
2.8. Indirect competitive ELISA
ꢀ
1
The microplate was coated with 100 mL of 1 mg mL of cyan-
ꢂ
traniliprole–OVA in coating buffer at 37 C for 3 h. The micro- 2.13. HPLC analysis
plate was washed with PBST three times aer coating, 50 mL of
Cyantraniliprole standards and pakchoi samples were analyzed
standard sample or analytes in PBSTG was added to each
7
with HPLC according to the procedure described by Schwarz
microplate well, followed by addition of 50 mL of mAb with a
8
and Sun with slight modication. An isocratic elution was used
ꢂ
dilution ratio of 1 : 20 000 in PBSTG. Aer incubated at 37 C for
.5 h, the microplate was washed again with PBST three times,
ꢀ1
with 80% aqueous acetonitrile at a ow rate of 1 mL min . The
0
retention time of cyantraniliprole was about 5 min. The residue
was dissolved in 2.0 mL of acetonitrile, and ltered with a 0.22
mm Millipore membrane. The ltered solution was used for
HPLC analysis. The HPLC results were compared with those
from icELISA to evaluate the reliability of the established
immunoassay.
and then an aliquot of 100 mL per well of goat anti-mouse IgG–
HRP with a ratio of 1 : 1000 diluted in PBSTG was added. Aer
ꢂ
incubated at 37 C for 0.5 h, the plate was washed again with
PBST three times, and then 100 mL per well of substrate solution
was added. Finally, the reaction was terminated by adding 50 mL
of the stop solution per well, and the absorbance was read with
the microplate reader at 492 nm. Data were calculated with
OriginPro 8.0 soware.
2.9. Specicity of antibody
The specicity of the mAb was evaluated by cross-reactivity (CR)
based on mass concentration with a set of structural analogs
through icELISA. The relative CR was calculated by the formula: CR
(%) ¼ (IC50 of cyantraniliprole/IC50 of other compounds) ꢁ 100.
2.10. Preparation of pakchoi samples
Field trials were carried out in Shangzhuang experimental eld
of China Agricultural University in 2013. Each experiment eld
2
consisted of three replicate plots with an area of 3 m each.
Cyantraniliprole (SC, 2.5%) was dissolved in water and sprayed
in the growing pakchoi using a knapsack sprayer at recom-
ꢀ
1
mended dosage (60 g a.i. ha ) with interval of 7 days between
each application for four times. The control was sprayed water
without any insecticide. Aer the application, the pakchoi was
sampled on day 7 aer spraying, and each pakchoi sample was
0
Fig. 2 Standard inhibition curve of cyantraniliprole by icELISA. B and
B were absorbance in the absence and presence of competitors,
respectively. Each point represents the mean of triplicate analyses.
approximate 200 g. At the same time, 11 pakchoi samples were Vertical bars indicate ꢃ standard deviations (SD) about the mean.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 35874–35881 | 35877

View Article Online
RSC Advances
Paper
Table 1 Assay cross-reactivity of cyantraniliprole and its analogs
ꢀ1
Chemical names
Structures
IC50 mg L
CR (%)
3
-Bromo-1-(3-chloro-2-pyridinyl)-N-(4-cyano-2-
methyl-6-((methylamino)carbonyl)phenyl)-1H-pyrazole-
1.57
100
5
-carboxamide (cyantraniliprole)
3
-Bromo-N-(4-chloro-2-methyl-6-((methylamino)-
carbonyl)phenyl)-1-(3-chloro-2-pyridinyl)-1H-pyrazole-5-
17.3
9
carboxamide (chlorantraniliprole)
0
3
(
-Iodo-N -(2-mesyl-1,1-dimethylethyl)-N-{4-(1,2,2,2-tetrauoro-1-
a
NI
<0.02
triuoromethyl]ethyl)-o-tolyl)}phthalamide (ubendiamide)
N-(2-(2-(2-Chlorobenzyl)hydrazinecarbonyl)-4-cyano-6-
methylphenyl)pivalamide
528
0.29
N-(4-Cyano-2-(2-(4-uorobenzyl)hydrazinecarbonyl)-6-
methylphenyl)pivalamide
6250
0.03
N-(2-(2-(2-Chlorobenzyl)hydrazinecarbonyl)-4-cyano-6-
methylphenyl)-3,3-dimethylbutanamide
a
NI
<0.02
35878 | RSC Adv., 2015, 5, 35874–35881
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
Table 1 (Contd.)
ꢀ1
Chemical names
Structures
IC50 mg L
CR (%)
0.09
N-(2-(2-Benzylhydrazinecarbonyl)-4-cyano-6-
methylphenyl)-3,3-dimethylbutanamide
1
5
1
840
080
620
N-(4-Cyano-2-(2-(2,4-dichlorobenzyl)hydrazinecarbonyl)-
0.04
0.10
6
-methylphenyl)-3,3-dimethylbutanamide
N-(4-Cyano-2-(2-(2-uorobenzyl)hydrazinecarbonyl)-6-
methylphenyl)-3,3-dimethylbutanamide
N-(4-Cyano-2-(2-(2,4-dichlorobenzyl)hydrazinecarbonyl)-
a
NI
<0.02
6
-methylphenyl)pivalamide
a
ꢀ1
No inhibition was observed up to 10 000 mg L of the analytes.
3.2. Optimization of icELISA
3. Results and discussion
In icELISA system, an IC50 value of the standard curve would be
affected by the concentrations of coating antigen and antibody.
The optimal concentrations of coating antigen and mAb were
screened by checkerboard titration. The coating antigen (1 mg
3
.1. Hapten design and synthesis, conjugation with proteins
It is known that hapten design plays a critical role on devel-
oping antibody for a small molecular weight compound. To
obtain a sensitive and selective cyantraniliprole antibody, the
aim of the hapten synthesis was to preserve the integrity of the
parent structure and to introduce a linker and functional
group so that the small molecule hapten could be conjugated
with carrier proteins. In order to preserve the structural
characteristics of cyantraniliprole at maximum extent, a
carboxylic propyl was introduced into cyantraniliprole, and
illuminated in synthetic route in Fig. 1. The UV-vis spectra
were used to monitor the conjugation of haptens and carrier
ꢀ1
ꢀ1
mL ) and puried mAb3B (0.14 mg mL ) were at a dilution
2
ratio of 1 : 100 000 and 1 : 20 000, respectively. The goat anti-
ꢀ1
mouse IgG–peroxidase conjugate (1 mg mL ) was at a dilu-
tion ratio of 1 : 1000. Fig. 2 showed a cyantraniliprole standard
curve in icELISA. A half maximal inhibition value (IC50) of the
ꢀ1
assay was 1.57 mg L , and a detection range (concentrations
ꢀ1
giving 80 and 20% B/B ) was 0.43–6.15 mg L
.
0
1
2,13
proteins.
The obtained spectra showed that the haptens 3.3. Antibody specicity
were successfully coupled with the carrier proteins. The molar
ratios of cyantraniliprole hapten to proteins were estimated to
be 15 : 1 and 8 : 1 for cyantraniliprole–BSA and cyan-
traniliprole–OVA, respectively.
2
To evaluate the specicity of mAb3B , chlorantraniliprole, u-
bendiamide and other cyantraniliprole analogues were used for
testing the cross-reactivity. The results summarized in Table 1
showed their structures, IC50 and the cross-reactivity with
cyantraniliprole. Flubendiamide, which was co-developed by
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 35874–35881 | 35879

View Article Online
RSC Advances
Paper
d
Nihon Nohyaku and Bayer Crop Science, also acted on insect Table 3 Determination of pakchoi samples by icELISA and HPLC
29
ryanodine receptors. However, due to distinct differences
between the chemical structures of ubendiamide and cyan-
traniliprole, the developed antibody as expected had no cross-
reactivity with ubendiamide. Chlorantraniliprole showed low Samples
cross-reactivity with cyantraniliprole (CR, 9.0%), because its
Concentrations of cyantraniliprole residues
ꢀ1
(ng g
)
a
icELISA
HPLC
a
b
1
1474.1 ꢃ 24.8
1437.0 ꢃ 52.2
4431.4 ꢃ 46.3
4670.0 ꢃ 57.1
680.0 ꢃ 18.2
1730.0 ꢃ 31.7
1587.0 ꢃ 63.4
4787.0 ꢃ 31.2
5039.0 ꢃ 43.6
1003.0 ꢃ 25.3
ND
structure was very similar to cyantraniliprole. The other chem-
icals showed very low or no cross-reactivity with cyan-
3
2
traniliprole. The hapten we had designed was very reasonable,
and the antibody specicity also ensured effective analysis of
cyantraniliprole residues in food and environmental samples.
4
5
6
c
–15
ND
a
b
Each sample was analyzed in triplicate. The data represented the
c
d
mean ꢃ SD. Not detected. Sample 1–4: pakchoi samples were
sprayed with cyantraniliprole. Sample 5–15: pakchoi samples were
bought from different local markets.
3
.4. Recoveries of cyantraniliprole in pakchoi and tap water
samples
The accuracy of an ELISA was commonly assessed by spiking
matrix samples with the target analyte, and then evaluating the
30
recovery. The average recoveries of cyantraniliprole with the
icELISA were 94–101% in both pakchoi and tap water (Table 2).
The good recoveries showed that the established icELISA was a
reliable and sensitive method to detect cyantraniliprole in
pakchoi and tap water samples.
practical analyses in the present study, pakchoi samples were
diluted by 250 or 500 fold. Therefore, if the residue of cyan-
traniliprole in leafy brassica vegetable samples reached 30 mg g
or higher, the samples should be diluted by more than 500
folds, which would have minimal background interference. It is
noteworthy that 50 fold or less dilution would be sufficient in
ꢀ1
3.5. Comparison of icELISA and HPLC for analysis of
analyses of most food and fruit samples to minimize the
cyantraniliprole in pakchoi samples
31,32
background interference.
The limit of detection of the icE-
ꢀ1
To be sure that icELISA was a reliable and sensitive method to LISA developed in the present study could reach 0.18 mg L
,
detect cyantraniliprole in pakchoi samples, HPLC had been which is 50 fold lower than the MRL of cyantraniliprole set by
employed for comparison (Table 3). The correlation coefficient PMRA. So the sensitivity of the assay was sufficient for screening
2
(
R ) between icELISA and HPLC results was 0.997. We found residual cyantraniliprole in real world samples. Overall, the
that the residual cyantraniliprole in most of the samples results demonstrated that the developed icELISA could be used
purchased from the local supermarkets were not detectable by to quickly and reliably monitor cyantraniliprole residues in
icELISA and HPLC. One reason may be that cyantraniliprole was environmental and agricultural samples.
8
degraded rapidly aer application, and another reason may be
that cyantraniliprole was lately registered in China and was
indeed not applied on the samples. The maximum residue limit
4
. Conclusion
ꢀ
1
(
MRL) of cyantraniliprole set by PMRA is 30 mg g on leafy To our knowledge, this is the rst paper on mAb-based icELISA
ꢀ1
11
brassica vegetables and 0.01 mg g in foods or fruits. In the for cyantraniliprole. The assay had low cross-reactivity with
chlorantraniliprole, and very low or no cross-reactivity with the
other cyantraniliprole analogues. The icELISA using mAb3B
2
ꢀ1
Table 2 Recovery of cyantraniliprole spiked to pakchoi samples and had an IC50 value of 1.57 mg L of cyantraniliprole, and a
ꢀ1
tap water
working range of 0.43–6.15 mg L based on 20–80% of inhibi-
tion. In addition, fortication tests of cyantraniliprole in pak-
choi and tap water samples by icELISA had average recoveries of
94–101%. Moreover, analyses of pakchoi samples sprayed with
cyantraniliprole and purchased from supermarkets suggested
that the result by icELISA agreed well with that by HPLC.
Overall, the results displayed that this new immunoassay could
be used for the quick and convenient determination of residual
cyantraniliprole in real world samples.
ꢀ
1
Concentrations (mg L
)
a
Samples
Pakchoi
Spiked
Detected
Recovery (%) ꢃ SD
b
0
ND
ND
2
5
1
2
4
0
1
2
5
8
1
50
00
000
000
000
234.3
485.0
944.0
2020.0
3840.0
ND
94 ꢃ 1.1
97 ꢃ 1.1
94 ꢃ 0.3
101 ꢃ 0.3
96 ꢃ 0.8
ND
94 ꢃ 1.0
98 ꢃ 2.8
98 ꢃ 3.4
101 ꢃ 0.9
97 ꢃ 1.2
Tap water
Conflict of interest
.25
.50
.00
.00
0.0
1.18
2.46
4.90
8.05
We declare that all authors have no nancial and personal
relationships with other people or organizations, which could
have improperly effect on our work. There also is no profes-
sional or personal interest of service or companies that could
inuence the position presented in, or the review of, the
9.69
a
b
Data were means ꢃ SD of three determinations. Not detected.
35880 | RSC Adv., 2015, 5, 35874–35881
This journal is © The Royal Society of Chemistry 2015

View Article Online
Paper
RSC Advances
manuscript entitled, “Development of a Monoclonal Antibody-
Based ELISA for the Detection of the Novel Insecticide
Cyantraniliprole”.
9 D. Kim, D. J. Brooks and H. Riedl, BioControl, 2006, 51, 465–
484.
10 L. A. Crampton, G. M. Loeb, K. A. Hoelmer and
M. P. Hoffmann, J. Insect Sci., 2010, 10, 36.
1
1 Pest Management Regulatory Agency (PMRA), (G/SPS/N/CAN/
01): Proposed Maximum Residue Limit: Cyantraniliprole
PMRL2013–42), 2013, URL http://www.hcsc.gc.ca/cps-spc/
Abbreviations
7
(
ELISA
IcELISA
HPLC
FBS
Enzyme-linked immunosorbent assay
Indirect competitive ELISA
High performance liquid chromatography
Fetal bovine serum
1-(3-Dimethylaminepropyl)-3-ethylcarbodiimide
4-Dimethylamino-pyridine
Dimethyl sulfoxide
Hypoxanthine, aminopterin, and thymidine
o-Phenylenediamine
Polyoxyethylenesorbitanmonolaurate
N-Hydroxysuccinimide
N-Iodosuccinimide
N,N-Dimethylformamide
N-Iodosuccinimide
Goat anti-mouse IgG
Phosphate-buffered saline
PBS with 0.1% (v/v) Tween-20
PBST containing 0.5% (w/v) gelatin
Cross-reactivity
pest/part/protect-proteger/food-nourriture/rccg-gcpcr-eng.php.
2 H. Zhao, T. Nan, G. Tan, W. Gao, Z. Cao, S. Sun, Z. Li, Q. X. Li
and B. Wang, Anal. Chim. Acta, 2011, 702, 102–108.
3 Y. L. Cui, K. C. Liu, C. Xu, F. Liu, Q. X. Li, S. Z. Liu and
B. M. Wang, Food Chem., 2014, 143, 293–299.
1
1
1
EDC
DMAP
DMSO
HAT
OPD
Tween-20
NHS
NIS
DMF
NIS
IgG–HRP
PBS
4 M. J. Moreno, A. Abad and A. Montoya, J. Agric. Food Chem.,
2001, 49, 72–78.
1
1
5 A. Dankwardt and B. Hock, Biotechnology, 1997, 35, 164–174.
6 E. P. Meulenberg, W. H. Mulder and P. G. Stoks, Environ. Sci.
Technol., 1995, 29, 553–561.
7 B. D. Hammock and S. J. Gee, Immunoanalysis for
Agrochemicals: Emerging Technologies, 1995, vol. 586, pp. 1–
1
19.
1
8 Z. Xu, Y. Shen, W. Zheng, R. C. Beier, G. Xie, J. Dong, J. Yang,
H. Wang, H. Lei and Z. She, Anal. Chem., 2010, 82, 9314–
9321.
PBST
PBSTG
CR
19 J. Y. Shim, Y. A. Kim, Y. T. Lee, B. D. Hammock and H. S. Lee,
J. Agric. Food Chem., 2010, 58, 241–247.
2
2
2
0 Q. Zhang, L. Wang, K. C. An, Q. Sun, B. Hu, J. Wang and
F. Liu, Anal. Chim. Acta, 2007, 596, 303–311.
1 L. Q. Liu, H. Kuang, C. F. Peng, L. B. Wang and C. L. Xu, Anal.
Methods, 2014, 6, 4228–4233.
2 L. Luo, Z. L. Xu, J. Y. Yang, Z. L. Xiao, Y. J. Li, R. C. Beier,
Y. M. Sun, H. T. Lei, H. Wang and Y. D. Shen, J. Agric. Food
Chem., 2014, 62, 12299–12308.
Acknowledgements
Preparation of uorescent ligand was supported by the National
S &T Pillar Program of China, no. 2012BAK25B03, the National
Natural Science Foundation of China (no. 21172256) and the 23 J. Wu, Y. D. Shen, H. T. Lei, Y. M. Sun, J. Y. Yang, Z. L. Xiao,
National Basic Research Program of China (no. 2010CB126104).
H. Wang and Z. L. Xu, J. Agric. Food Chem., 2014, 62, 7078–
7084.
2
4 J. Ou, X. Zhu, L. Wang, C. Xu, F. Liu, L. Ren, X. Xu, Y. Wang,
C. Rui and S. Liu, J. Agric. Food Chem., 2012, 60, 10942–
10951.
References
1
I. Ishaaya and D. Degheele, Insecticides with novel modes of
action: mechanism and application, Springer-Verlag, Berlin, 25 X. Zhang, A. Liu, Y. Zhao, L. Xiong and Z. Li, Chem. Res. Chin.
Germany, 1998, pp. 260–281. Univ., 2013, 29, 1134–1139.
S. P. Foster, I. Denholm, J. L. Rison, H. E. Portillo, 26 G. P. Lahm, T. P. Selby, J. H. Freudenberger, T. M. Stevenson,
2
J. Margaritopoulis and R. Slater, Pest Manage. Sci., 2012,
8, 629–633.
B. J. Myers, G. Seburyamo, B. K. Smith, L. Flexner, C. E. Clark
and D. Cordova, Bioorg. Med. Chem. Lett., 2005, 15, 4898–4906.
6
3
4
D. B. Sattelle, D. Cordova and T. R. Cheek, Invertebr. 27 S. R. Pingali, M. Madhav and B. S. Jursic, Tetrahedron Lett.,
Neurosci., 2008, 8, 107–119. 2010, 51, 1383–1385.
J. A. Wiles, I. B. Annan, H. E. Portillo, J. L. Rison, A. Dinter 28 Y. Zhao, Y. Li, X. Ou, P. Zhang, Z. Huang, F. Bi, R. Huang and
and N. M. Frost, Association Française de Protection des
Plantes (AFPP), 2011, 25–27, 698–705.
B. S. Chai, X. M. He, J. F. Wang, Z. N. Li and C. L. Liu,
Agrochemicals, 2010, 49, 167–169.
Q. Wang, J. Agric. Food Chem., 2008, 56, 10176–10182.
29 T. Masaki, N. Yasokawa, M. Tohnishi, T. Nishimatsu,
K. Tsubata and K. Inoue, Mol. Pharmacol., 2006, 69, 1733–
1739.
5
6
7
A. L. Jacobson and G. G. Kennedy, Crop Prot., 2011, 30, 512– 30 Y. Zhang, A. H. Gao, B. Liu, W. Sheng, C. Tan, M. Yuan and
15.
S. Wang, J. Environ. Sci. Health, Part B, 2013, 48, 1–8.
T. Schwarz, T. A. Snow, C. J. Santee, C. C. Mulligan, T. Class, 31 M. Qian, H. Zhang, L. Wu, F. Liu, X. Wang, H. He and
5
M. P. Wadsley and S. C. Nanita, J. Agric. Food Chem., 2010, 59,
14–821.
J. Sun, N. Feng, C. Tang and D. Qin, Bull. Environ. Contam.
Toxicol., 2012, 89, 845–852.
Z. Chen, Chin. J. Anal. Chem., 2010, 5, 702–706.
32 Z. Xu, H. Deng, X. Deng, J. Yang, Y. Jiang, D. Zeng, F. Huang,
Y. Shen, H. Lei, H. Wang and Y. Sun, Food Chem., 2012, 131,
1569–1576.
8
8
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 35874–35881 | 35881