Development of a sensitive indirect competitive enzyme-linked immunosorbent assay based on the monoclonal antibody for the detection of benzothiostrobin residue

Article abstract of DOI:10.1039/c4ra01845a

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on monoclonal antibodies (MAbs) for benzothiostrobin has been developed. The hapten of benzothiostrobin was synthesized and conjugated to bovine serum albumin and ovalbumin to generate an immunogen and a coating antigen. The immunogen was used to immunize BALB/c mice, resulting in anti-benzothiostrobin MAb. Under optimal conditions, the half maximal inhibition concentration (IC₅₀) and the limit of detection (IC₁₀) of the developed ic-ELISA were 7.55 and 0.428 μg L^-1, respectively. The cross-reactivity (CR) was less than 0.05% for the tested structural analogues and regarded as negligible except for pyraclostrobin, which exhibited a CR of 0.34%. The recoveries of benzothiostrobin ranged from 80.43 to 113.83% in environmental and agricultural samples, respectively, which conformed to the requirements for residue detection. The amount of benzothiostrobin detected by ic-ELISA in the samples was significantly (R² = 0.9894, y = 1.0867x + 0.0318) correlated with that detected by high-performance liquid chromatography (HPLC). The current study indicates that the established ic-ELISA is a potentially useful tool for detecting benzothiostrobin in environmental and agricultural samples. The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c4ra01845a

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Development of a sensitive indirect competitive
enzyme-linked immunosorbent assay based on the
monoclonal antibody for the detection of
benzothiostrobin residue†
Cite this: RSC Adv., 2014, 4, 24406
*
Yulong Yuan,‡ Xiude Hua,‡ Ming Li, Wei Yin, Haiyan Shi and Minghua Wang
An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on monoclonal antibodies
(MAbs) for benzothiostrobin has been developed. The hapten of benzothiostrobin was synthesized and
conjugated to bovine serum albumin and ovalbumin to generate an immunogen and a coating antigen.
The immunogen was used to immunize BALB/c mice, resulting in anti-benzothiostrobin MAb. Under
optimal conditions, the half maximal inhibition concentration (IC50) and the limit of detection (IC10) of
the developed ic-ELISA were 7.55 and 0.428 mg Lꢀ1, respectively. The cross-reactivity (CR) was less than
0.05% for the tested structural analogues and regarded as negligible except for pyraclostrobin, which
exhibited a CR of 0.34%. The recoveries of benzothiostrobin ranged from 80.43 to 113.83% in
environmental and agricultural samples, respectively, which conformed to the requirements for residue
Received 3rd March 2014
Accepted 15th May 2014
detection. The amount of benzothiostrobin detected by ic-ELISA in the samples was significantly (R2
¼
0.9894, 1.0867x 0.0318) correlated with that detected by high-performance liquid
y
¼
+
DOI: 10.1039/c4ra01845a
chromatography (HPLC). The current study indicates that the established ic-ELISA is a potentially useful
tool for detecting benzothiostrobin in environmental and agricultural samples.
www.rsc.org/advances
sensitive, and economical method for detecting benzothios-
trobin in agricultural produce and the environment as a tech-
1. Introduction
nical support is imperative and important.
Strobilurin fungicide belongs to a new class of fungicides that
exhibits broad-spectrum, efficient and environmentally friendly
features. This new class of fungicides has attracted considerable
attention from researchers. Benzothiostrobin, which is a
fungicide developed by the Central China Normal University, is
a novel strobilurin fungicide that exhibits highly efficient
control of most fungal diseases in crops,1 and the patents of
preparing and using benzothiostrobin have been applied in
China, United States, Europe, and worldwide (Pub. no.: CN
102302012 B, CN 101379967 A, CN 101268780 B, US 2010/
0292285 A1, 09741682.0, WO 2007/073637 A1, WO 2009/135407
A1). To the best of our knowledge, benzothiostrobin was
transferred to Jiangsu Seven Continent Green Chemical Co.,
Ltd. in 2010. Therefore, benzothiostrobin, at least in China, has
potential prospective applications. In order to keep its potential
residual risk under control, the development of a rapid,
High-performance liquid chromatography (HPLC) has been
used successfully for the detection of benzothiostrobin and
other pesticides,2 which is characterized by low limits of
detection (LOD) as well as high precision and sensitivity.
However, the analytical methods are costly, time-consuming,
and not suitable for the analysis of large numbers of samples.3,4
During the past decade, many enzyme-linked immunosorbent
assays (ELISAs) have been published for the analysis of residues
for a broad variety of pesticides in different types of food
samples,5–11 and several immunochemical methodologies have
been approved to analyze chemical residues in food, feed and
environmental samples. In comparison, ELISAs provide a fast,
sensitive, cost-effective, and selective method for the detection
of pesticide residues. Several immunoassays for strobilurin
fungicides have been reported.12–18 However, studies on anti-
bodies and the development of immunoassays for benzothios-
trobin residue have not been published.
Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural
University, Jiangsu Key Laboratory of Pesticide Science, Key Laboratory of Integrated
Management of Crop Diseases and Pests, Ministry of Education, Nanjing 210095,
People's Republic of China. E-mail: wangmha@njau.edu.cn; Fax: +86 25 84395479;
Tel: +86 25 84395479
In this paper, an indirect competitive ELISA (ic-ELISA)
method was developed based on monoclonal antibodies for the
detection of benzothiostrobin. Due to this method's high
sensitivity and accuracy, ic-ELISA provides an alternate method
for the detection of benzothiostrobin in environmental and
agricultural samples.
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra01845a
‡ Both authors contributed equally to this paper.
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Na2SO4 and concentrated under reduced pressure to obtain a
yellow oil, which was puried on a silica gel column eluted
using 150 mL of methanol–dichloromethane (1 : 50 v/v) and 200
mL of methanol–dichloromethane (1 : 30 v/v).19 The second
fraction was collected and concentrated to obtain hapten,
which was characterized by ESI-MS and NMR: ESI-MS, m/z: 346
[M + H+] and 368 [M + Na+]; 1H NMR (400 MHz, DMSO) d: 3.79
(s, 2H, CH2COO), 3.86 (s, 3H, OCH3), 4.67 (s, 2H, SCH2), 7.01
(dd, 1H, ArH), 7.32–7.22 (m, 3H, ArH), 7.47 (d, 1H, ArH), 7.52
(m, 1H, ArH), 7.88 (m, 1H, ArH), 12.52 (s, 1H, COOH).
2. Materials and methods
2.1. Materials and instruments
Benzothiostrobin (99.02%) was obtained from the Central China
Normal University (Wuhan, China). The pesticide standards used
for cross-reactivity studies were purchased from Dr Ehrenstorfer
GmbH (Germany). Bovine serum albumin (BSA), ovalbumin
(OVA), complete and incomplete Freund's adjuvants, N-hydroxy-
succinimide (NHS), N,N0-dicyclohexylcarbodiimide (DCC), o-
phenylenediamine (OPD), and polyoxyethylene sorbitan mono-
laurate (Tween-20) were purchased from Sigma-Aldrich Chemical
Co. (St. Louis, MO). Horseradish peroxidase (HRP)-labeled goat
anti-mouse IgG was purchased from DingGuo ChangSheng
Biotechnology Co. (Beijing, China). All the other reagents were of
analytical grade. The BALB/c mice were purchased from the
Center of Comparative Medicine of Yangzhou University
(Yangzhou, China). All the animal studies were performed in
accordance with institutional guidelines.
Mass spectral (MS) data were obtained with an HPLC-QTOF
instrument (Bruker, Germany). Nuclear magnetic resonance
(NMR) spectra were recorded on a DRX500 spectrometer
(Bruker, Germany). Ultraviolet-visible (UV-Vis) spectra were
obtained on a UV-2550 spectrophotometer (Shimadzu, Japan).
The 96-well transparent microplates were purchased from
Nunc (Roskilde, Denmark). The absorbances were read with an
I-mark microtiter plate reader (Bio-RAD, USA) at 490 nm, and
the ELISA plates were washed with a Wellwash Plus (Thermo,
USA). Benzothiostrobin was detected using Agilent 1200 HPLC
(Agilent, USA). The antibody was freeze-dried using a vacuum
freeze-drying machine (Thermo Savant, USA). The protein A
column was purchased from the Pall Corporation (Pall, USA).
2.4. Preparation of hapten–protein conjugates
The hapten was conjugated with BSA via the carbodiimide
method to produce an immunogen and conjugated with OVA
via the mixed anhydride method to produce a coating antigen.20
The conjugates were dialyzed in PBS over 72 h at 4 ꢁC and stored
at ꢀ20 ꢁC. The formations of conjugates were conrmed by
UV-Vis spectroscopy, and the hapten densities (the number of
hapten molecules per molecule of protein) of the conjugates
were estimated according to the following formula:
KAx ¼ 3Ax/(rx/Mx),
KBx ¼ 3Bx/(rx/Mx)
Ca/Cb ¼ (3Ca ꢂ KBb ꢀ 3Cb ꢂ KBa)/(3Cb ꢂ KAa ꢀ 3Ca ꢂ KAb)
where A, B, and C represent the hapten, protein and conjuga-
tion, respectively; x represents a and b; 3Aa, 3Ba and 3Ca are their
ultraviolet absorbances at the characteristic absorption wave-
length of hapten and 3Ab, 3Bb and 3Cb are their ultraviolet
absorbances at the characteristic absorption wavelength of the
protein; r is the concentration; and M is the molar mass.21,22
2.2. Buffers and solutions
Phosphate-buffered saline (PBS, 0.01 mol Lꢀ1, pH 7.4), carbonate-
buffered saline (CBS, 0.05 mol Lꢀ1, pH 9.6), and phosphate-
buffered saline containing 0.05% Tween-20 (PBST) were used. The
tetramethylbenzidine (TMB) solution contained 0.4 mmol Lꢀ1
TMB and 3 mmol Lꢀ1 H2O2 in a citrate buffer (pH 5.0).
2.5. Immunization and monoclonal antibody preparation
Six-week-old female BALB/c mice were immunized with
immunogen via an intraperitoneal injection according to the
method described by Kishiro et al.23 The dosage of the rst
immunogen for each mouse was 100 mg (7.93 mg mLꢀ1) dis-
solved in physiological saline and emulsied with an equal
volume of Freund's complete adjuvant. Aer 3 weeks, four
subsequent injections were given at 2-week intervals using
immunogen emulsied with incomplete Freund's adjuvant. The
antisera were obtained from the tail vein of the mouse one week
aer the immunization, and the sera were tested by ic-ELISA to
determine their ability to conjugate with benzothiostrobin. The
mouse that exhibited the strongest response was used for the
peritoneal cavity injections of 200 mg of immunogen in PBS 1
week aer the last immunization. Three days aer the booster
injection, cell fusion was performed according to Nowinski
et al.24 Mouse spleen lymphocytes were fused with SP2/
0 myeloma cells in a 5 : 1 ratio. The fused cells were cultured in
a hypoxanthine–aminopterin–thymidine (HAT) medium at
2.3. Hapten synthesis
The synthesis route of the hapten is shown in Fig. 1. Here, 8.02 g
of benzothiostrobin (0.02 mol) was added to 10 mL of 10 mol
Lꢀ1 LiOH (0.1 mol), and the mixture was stirred at room
temperature for 4 h. Then, 30 mL of water was added to the
mixture followed by extraction with ethyl acetate. The aqueous
phase was adjusted to pH 3.0 with 10% HCl and extracted with
ethyl acetate, and the organic phase was dried over anhydrous
ꢁ
37 C in an atmosphere of 5% CO2. Half of the media in the
wells were replaced by fresh HAT media every third day. Four-
teen days aer cell fusion, the HAT media were changed to HT
media. Culture supernatants were screened for their ability to
Fig. 1 Synthetic route of the benzothiostrobin hapten.
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recognize benzothiostrobin, and hybridoma cells in the ELISA
positive wells were cloned using the limiting dilution method.
Stable antibody-producing clones were expanded. Ascites
obtained from BALB/c mice were puried using a protein A
column and stored at ꢀ20 ꢁC aer freeze-drying. The subtyping
of MAb was identied by “mouse monoclonal antibody isotyp-
ing reagents” (Sigma).
2.9. Analysis of spiked samples
To evaluate the accuracy and precision of the immunoassays,
the recoveries of the spiked samples were studied by ELISA. Ten
grams of the blank samples (including river water, soil, rice,
pear, or tomato sample) were spiked with benzothiostrobin at
0.01, 0.05, or 0.5 mg kgꢀ1 and stored overnight. The river water
samples were mixed with the same volume of PBS containing
10% methanol and analyzed by ELISA. Other samples were
extracted twice by sonication in 20 mL of methanol for 10 min
and centrifuged for 10 min at 4000 rpm.28 Aer dilution at
appropriate multiple steps, the solutions were analyzed via the
immunoassays. The spiked recoveries were used to conrm the
accuracy of ic-ELISA.
The matrix interferences of the samples were analyzed by
testing 2 to 20-fold dilutions with PBS containing methanol.29
The matrix interference was determined by comparing the
benzothiostrobin standard curve prepared in matrix-free PBS
with calibration curves prepared with a series of diluted matrix
extracts.
2.6. Immunoassay procedure
ELISA was performed on 96-well polystyrene microplates. The
coating antigen (benzothiostrobin–OVA) was diluted with CBS,
pipetted into the wells (100 mL per well), and incubated over-
night at 4 ꢁC. The plate was washed 5 times with PBST and
blocked by adding 1% OVA/PBS (200 mL per well), followed by
incubation for 50 min at 37 ꢁC. Aer 5 repeated washings with
PBST, either the sample or the standard in the PBS containing
methanol (50 mL per well) was added, followed by the addition
of the diluted antibody (50 mL per well, in PBS) and incubated
for 1 h at 37 ꢁC. Aer an additional wash, the diluted goat anti-
mouse IgG-HRP (1 : 10 000 in PBS, 100 mL per well) was
dispensed into each well and incubated for 1 h at 37 ꢁC. Then,
the plates were washed again, and the substrate solution was
added (100 mL per well). The reaction was terminated with
sulfuric acid (2 mol Lꢀ1, 50 mL per well) aer 15 min of incu-
bation. The absorbance was measured at 490 nm.
2.10. Practical application of the immunoassays
Soil and pear (1, 3, 7, 14 days aer sprayed) were collected from
farms where benzothiostrobin had been used in Nanjing,
China. The concentrations of benzothiostrobin in the samples
were simultaneously analyzed by ELISA and HPLC to evaluate
the correlation between the two methods. For ELISA, the
extraction and analysis of the samples were performed as
described above. For HPLC, the samples were extracted twice
by sonicating in 20 mL of acetonitrile for 10 min and centri-
fuging for 10 min at 4000 rpm. The supernatant was ltered
through anhydrous sodium sulfate and concentrated. Then,
the concentrates were diluted with 2 mL of acetonitrile and
detected by HPLC with SB-C18 column (250 mm ꢂ 4.6 mm i.d.)
at 230 nm, and acetonitrile–0.05% triuoroacetic acid
(65 : 35, v/v) was used as the mobile phase at a ow rate of
2.7. Assay optimization
The optimal concentrations of coating antigen and antibody
were conrmed using the two-dimensional checkerboard
method.25 The experimental parameters including the organic
solvent, ionic strength, and buffer pH value were sequentially
studied to improve the sensitivity of the immunoassay.
Competitive curves were run in PBS solutions containing
different concentrations of methanol (from 5 to 30%, v/v) and
Na+ (0.1, 0.2, 0.3, 0.4 and 0.5 mol Lꢀ1) as well as with various pH
values (5.5, 6.5, 7.5 and 8.5), and the evaluations of the
parameters were based on the Amax/IC50, IC50 and the squared
coefficients of correlation (R2) of the linear equation.26
0.8 mL minꢀ1
.
2
A standard curve for benzothiostrobin was obtained under
the optimum conditions by plotting the percent binding
(% B/B0) as a function of the concentration of benzothiostrobin
(C). The % B/B0 was calculated using the following formula:
% B/B0 ¼ [(Ax ꢀ Amin)/(Amax ꢀ Amin)] ꢂ 100
where Ax is the absorbance of the sample, Amax is the absor-
bance in the absence of the analyte, and Amin is the absorbance
of the background.
2.8. Cross-reactions
The cross-reactivity (CR) of ic-ELISA was studied by detecting
the analogues of benzothiostrobin.27 The CR values were
calculated with the following formula:
CR% ¼ (IC50 of benzothiostrobin/IC50 of analogue) ꢂ 100
24408 | RSC Adv., 2014, 4, 24406–24411
Fig. 2 Standard curve for benzothiostrobin by ELISA.
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structure of the obtained hapten was similar to benzothios-
trobin, and the hapten contained a carboxyl group that could be
conjugated to a carrier protein. In addition, the next study
conrmed that the obtained hapten could be used to prepare
specic anti-benzothiostrobin antibodies. The UV-Vis spectra
3. Results and discussion
3.1. Identication of articial antigens and coupling ratio
Under alkaline conditions, the ethylene bond of the reactant
dissociated. With the identication by ESI-MS and NMR, the
Table 1 Cross-reactivity of a set of analogs related to benzothiostrobin by ELISA
Compound
Structure
IC50 (mg Lꢀ1
)
CR (%)
100
Benzothiostrobin
0.00755
Pyraclostrobin
Azoxystrobin
2.25
>100
>100
0.34
<0.05
<0.05
Kresoxim-methyl
Picoxystrobin
>100
>100
<0.05
<0.05
Chloropiperidine ester
Fluoxastrobin
>100
>100
<0.05
<0.05
Fenaminstrobin
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indicated qualitative differences between the hapten, carrier samples, a 5-fold dilution (soil and pear) and 10-fold dilution
protein and conjugates, especially at 280 nm, and conrmed (tomato and rice) were selected for subsequent immunoassays.
that the carrier protein and hapten had been successfully
coupled. The molar ratios (hapten/protein) were 17 : 1 and 8 : 1
for the immunogen and coating antigen, respectively.
3.7. Analysis of spiked samples
As shown in Table 2, the immunoassay method had acceptable
recoveries from 83.4 to 115.6%, and the relative standard
deviations (RSDs) ranged from 1.8 to 6.8%. The test results were
conrmed by HPLC, and good correlations between the HPLC
results and the immunoassay results were obtained. These
results indicated that the precision of ic-ELISA met the
requirement of detecting residual benzothiostrobin in the
samples.
3.2. Subtyping of MAb
Two cell lines that can stably produce MAbs against benzo-
thiostrobin were obtained and named 4E8 and 2B4. By deter-
mining the immunoglobulin subclass, the results indicated
that 4E8 and 2B4 were of the lgG2a and lgM subclasses with a
kappa light chain, respectively. In this study, 4E8 exhibited
higher sensitivity than 2B4 and was used in further studies.
3.3. Optimization of immunoassays
3.8. Analysis of authentic samples
The optimal concentrations of the coating antigen and antibody
were 500 and 35 mg Lꢀ1, respectively. For solvent optimization,
methanol was used to improve the solubility of the analyte and
evaluate its effect on the ELISA method of benzothiostrobin.
With the methanol concentrations being 5%, the ic-ELISA
method showed the highest Amax/IC50 and lowest IC50. The ionic
strength and pH of the buffer also affected the sensitivity of ic-
ELISA; when the PBS buffer contained 0.3 mol Lꢀ1 Na+ at pH 6.5,
the assay exhibited the lowest IC50 and highest sensitivity.
Therefore, the optimum parameters included 5% methanol and
0.3 mol Lꢀ1 Na+ at pH 6.5, resulting in ELISA with the lowest
Table 3 shows the results from the established ELISA for ben-
zothiostrobin-positive soil and pear samples in Nanjing, China.
According to the results (from 70 to 1490 mg Lꢀ1), the residue in
the soil and pear samples progressively declined over time. The
results of the immunoassays and HPLC for authentic samples
exhibited good correlations (R2 ¼ 0.9894, y ¼ 1.0867x + 0.0318)
Table 2 Recovery of benzothiostrobin in spiked samples (n ¼ 3)
Spiked concentration
Average recovery
ꢃ SD (%)
Sample
(mg Lꢀ1 or mg kgꢀ1
)
RSD%
IC50 value and maximum Amax/IC50
.
River water
0.01
0.05
0.5
0.01
0.05
0.5
0.01
0.05
0.5
0.01
0.05
0.5
85.3 ꢃ 2.2
93.5 ꢃ 1.7
100.2 ꢃ 3.8
89.7 ꢃ 1.2
89.4 ꢃ 3.6
95.5 ꢃ 3.2
113.1 ꢃ 6.3
107.4 ꢃ 2.8
85.3 ꢃ 3.5
93.3 ꢃ 3.2
95.6 ꢃ 1.5
100.1 ꢃ 2.4
115.6 ꢃ 7.9
109.8 ꢃ 5.5
83.4 ꢃ 3.2
2.6
1.8
3.8
1.3
4.0
3.4
5.6
2.6
4.1
3.4
1.6
2.4
6.8
5.0
3.8
3.4. Sensitivities
Under the optimum conditions, the calibration curve was
obtained using the relationship between the percent binding (%
B/B0) and the concentration of benzothiostrobin shown in
Fig. 2. The assay indicated that IC50 was 7.55 mg Lꢀ1 and LOD
(IC10) was 0.428 mg Lꢀ1. The linear range (IC10–IC90) was 0.428–
54 mg Lꢀ1. In a published article, the linear range was 0.1–10 mg
Lꢀ1 for benzothiostrobin by HPLC analysis.2 Comparing the two
methods, ELISA was more sensitive than HPLC.
Soil
Rice
Pear
Tomato
0.01
0.05
0.5
3.5. Specicity
Table 1 shows the CRs result for the analogues of benzothios-
trobin. The highest cross-reactivity was observed for pyraclos-
trobin (0.34%). The antibody exhibited negligible cross-
reactivity (CR% < 0.05%) for the other analogues. Therefore, the
assays exhibited strong specicity for benzothiostrobin.
Table 3 Comparison of benzothiostrobin residues between the ELISA
and HPLC in authentic samples (n ¼ 3)
ELISA
HPLC
3.6. Matrix effects
Mean
RSD
(%)
Mean
RSD
(%)
The results of the matrix effects study indicated that different
sample matrices had different effects on the sensitivity of the
immunoassays. The matrices of the rice and tomato samples
had a substantial impact, and their effects were reduced to
acceptable levels by a 10-fold dilution. The matrices of the pear
and soil samples exhibited a small impact. For a 5-fold dilution
of the pear and soil samples, the interference had no effect on
the sensitivity of the immunoassays. Because too much dilution
would eventually reduce the limit of quantication of the
Sample
Soil
(mg kgꢀ1
)
(mg kgꢀ1
)
1 day
1.49
1.12
0.78
0.64
0.81
0.63
0.45
0.07
3.1
2.9
3.3
3.2
5.1
6.1
4.8
5.4
1.37
1.01
0.67
0.57
0.70
0.55
0.31
0.11
2.5
2.6
2.7
2.6
4.2
4.5
5.0
4.1
3 days
7 days
14 days
1 day
3 days
7 days
14 days
Pear
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´
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Acknowledgements
This work was supported by the Special Fund for Agro-scientic
Research in the Public Interest (no. 201203022) and the Youth
Innovation Fund in Science & Technology of Nanjing Agricul-
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