Rational hapten design to produce high-quality antibodies against carbamate pesticides and development of immunochromatographic assays for simultaneous pesticide screening

Article abstract of DOI:10.1016/j.jhazmat.2021.125241

Carbamate pesticides (CPs) are the most used pesticides in agricultural production and pest control. In this study, carbofuran, isoprocarb and carbaryl were employed as models, and a general hapten strategy based on carbamate moiety recognition was propos

Full text of DOI:10.1016/j.jhazmat.2021.125241

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Contents lists available at ScienceDirect
Journal of Hazardous Materials
journal homepage: www.elsevier.com/locate/jhazmat
Rational hapten design to produce high-quality antibodies against
carbamate pesticides and development of immunochromatographic assays
for simultaneous pesticide screening
,
,
,
Zi-Jian Chena 1, Hui-Ling Wua 1, Zhi-Li Xiaoa *, Hui-Jun Fua, Yu-Dong Shena, Lin Luoa,
Hong Wanga, Hong-Tao Leia, Surat Hongsibsongb, Zhen-Lin Xua
,
*
a Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, China
b Research Institute for Health Science, Chiang Mai University, Chiang Mai 50200, Thailand
A R T I C L E I N F O
Editor: Dr. S. Nan
A B S T R A C T
Carbamate pesticides (CPs) are the most used pesticides in agricultural production and pest control. In this study,
carbofuran, isoprocarb and carbaryl were employed as models, and a general hapten strategy based on carba-
mate moiety recognition was proposed. Molecular modeling of the three-dimensional (3D) structure and surface
electrostatic potential of the CPs indicated that the amide group formed by conjugation significantly influenced
recognition by antibodies. The proposed strategy was used to obtain three sensitive and specific monoclonal
antibodies (mAbs) with IC50 values of 1.4 ng/mL, 8.4 ng/mL and 13.8 ng/mL for carbofuran, isoprocarb and
carbaryl, respectively. Negligible cross-reactivity (%) with analogs was observed, except for fenobucarb (84.6%)
for isoprocarb. The obtained antibodies were used to develop an immunochromatographic assay (ICA) to
simultaneously and quantitatively detect the three CPs. A strip reader was used to determine the limits of
quantitation (LOQs) as 0.05 ng/mL (carbofuran), 31.3 ng/mL (isoprocarb) and 31.3 ng/mL (carbaryl). The re-
coveries of cucumber and Chinese cabbage samples ranged from 76% to 111%, with CVs from 1.3% to 10.6%,
indicating good potential for the rapid simultaneous detection of multiple pesticide residues in a large batch of
samples.
Keywords:
Carbamate pesticide
Hapten design
Molecular modeling
Monoclonal antibody
Immunochromatography assay
1. Introduction
1000 ng/g in vegetables, respectively (GB, 2763—2019). Strict MRLs for
these three CPs have also been set by the EU (1 ng/g~10 ng/g; EU Plant
Carbamate pesticides (CPs) can inhibit the enzyme acetylcholines-
terase (AChE) and are therefore widely used to kill insects (Guo et al.,
2013; Zhou et al., 2018). However, the improper and excessive CP use
leaves residues on foods and in aqueous environments, which is a serious
problem. CPs can inhibit AChE activity in the human central and pe-
ripheral nervous systems (Yan et al., 2019) and the resulting accumu-
lation of the neurotransmitter acetylcholine in the body (Wang et al.,
2016) leads to acute poisoning. Moreover, the long-term intake of foods
with pesticide residue levels in excess of the recommended standards
can cause chronic poisoning and even poses a cancer risk (Yang et al.,
2018). Therefore, it is necessary to monitor CP residues in foods. Gov-
ernments and organizations worldwide have set low maximum residue
limits (MRLs) for CPs. The Chinese MRLs for the most commonly used
CPs, carbofuran, isoproacrb and carbaryl, are 20 ng/g, 500 ng/g and
Pesticides database), the USA (0.1 μg/g~215 μg/g; States Environ-
mental Protection Agency (EPA)) and the Codex Alimentarius Com-
mission (10 ng/g~30
index).
μg/g; Codex Alimentarius Commission pesticide
Several methods have been developed for CP detection, including
HPLC-MS (Shi et al., 2016), GC-MS (Tripathy et al., 2019) and the use of
electrochemical sensors (Costa et al., 2017) and biosensors (Campanella
et al., 2007; Mavrikou et al., 2008; Song et al., 2016). Instrumental
methods can be used to detect carbamate pesticides with high sensi-
tivity, accuracy and repeatability. However, strict standards for sample
pretreatment and operation are required for these methods. Instru-
mental methods also have disadvantages, such as high time consump-
tion, nonportability, high cost and low throughput, which make it
difficult to meet the requirements for monitoring CPs. Immunoassays
* Corresponding authors at: Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Guangzhou 510642, China.
E-mail addresses: scauxzl@scau.edu.cn (Z.-L. Xiao), xzlin@scau.edu.cn (Z.-L. Xu).
1
Equal contribution.
https://doi.org/10.1016/j.jhazmat.2021.125241
Received 3 November 2020; Received in revised form 12 January 2021; Accepted 23 January 2021
Available online 27 January 2021
0304-3894/© 2021 Elsevier B.V. All rights reserved.

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Z.-J. Chen et al.
Journal of Hazardous Materials 412 (2021) 125241
Fig. 1. The hapten synthetic routes for the carbamate pesticide.
are an alternative technique that has been widely used for CP detection
and is rapid, cost-effective and facile, with a high throughput (Abad and
Montoya, 1994; Lan et al., 2020; Liu et al., 2019; Yao et al., 2017).
Hapten design is a key element of this technique and determines the
sensitivity and specificity of the prepared antibody and the competi-
tiveness of the coating antigen. The main strategy used in the design of
CP haptens has been to introduce a carboxylic spacer arm into the
methyl group of the carbamate moiety (Abad et al., 1998; Yao et al.,
2017) to maintain the molecular conformation and charge distribution
of the original CP. These haptens are typically used to prepare immu-
nogens. Hapten design for coating antigens is another key influence
factor for the sensitivity of immunoassays. Heterologous coatings have
been effectively used in many studies to increase the sensitivity of im-
munoassays. Several heterologous hapten designs for CPs have been
reported, including modification of the main characteristic structure,
the carbamate moiety, and the spacer arm (Abad et al., 1998, 1997a;
Dong et al., 2010). Heterologous haptens are conformationally different
from immunized haptens and are therefore less competitive with ana-
lytes, which increases the sensitivity of immunoassays. However, ho-
mologous coatings with higher titers also been used to detect some CPs
and have a higher sensitivity than heterologous coatings (Yao et al.,
2017; Zhang et al., 2019; Abad and Montoya, 1994; Moreno et al., 2001;
Abad et al., 1997b; Sun et al., 2009).
In this study, we employed the most used CPs, including carbofuran,
isoprocarb and carbaryl, as models to successfully design a general
hapten strategy for producing high-quality monoclonal antibodies.
Computer-assisted molecular modeling was used to determine the
recognition mechanism between antibodies and antigens. The three
obtained antibodies were used to develop easy-to-use immunochroma-
tographic assays (ICAs), achieving simultaneous and quantitative
detection in real samples.
2

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2. Materials and methods
J = 5.7 Hz, 1 H), 7.00 (d, J = 7.2 Hz, 1 H), 6.84 (d, J = 8.0 Hz, 1 H),
6.74 (t, J = 7.7 Hz, 1 H), 3.01 (m, 4 H), 2.50 (d, J = 1.5 Hz, 3 H), 2.20
(t, J = 7.4 Hz, 2 H), 1.50 (m, 13 H), 1.43 (dd, J = 14.8, 7.3 Hz, 2 H),
1.39 (s, 6 H), 1.30 (dd, J = 15.2, 8.0 Hz, 2 H).
2.1. Reagents and animals
Standards of carbofuran, isoprocarb, carbaryl, carbofuran-3-
hydroxy, carbosulfan, benfuracarb, metolcarb and fenobucarb were
supplied by TMRM Co. Ltd. (Beijing, China). Benzofuranol, 2-isopropyl-
phenol, 1-naphthol, ethyl bromoacetate, ethyl 4-bromobutyrate, 4-
nitrophenyl chloroformate, 6-aminocaproic acid and 1-naphthoxyacetic
acid were purchased from Heowns Biochemical Technology Co., Ltd.
2.3.2. Hapten-B, 2-((2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)oxy)acetic
acid
Benzofuranol (3.2 g, 20 mmol) and K2CO3 (2 g) were added to 10 mL
DMF and stirred at 70 ◦C. Ethyl bromoacetate (4.5 mL, 40 mmol) was
then added to the mixture, which was stirred at 70 ◦C for 6 h. After the
reaction was completed, the mixture was added to ethyl acetate and
washed with brine to remove K2CO3 and DMF. The organic phase was
evaporated, and an LiOH solution (1.6 g, 10 mL) was added to the
resulting mixture. The mixture was stirred for 1 h. The aqueous phase
was washed with ethyl acetate three times to remove the unhydrolyzed
product, and HCl was used to adjust the pH of the mixture to 4–5. The
crude product was subsequently extracted by ethyl acetate. Ethyl acetate
was then evaporated to obtain the final product. The following results
were obtained from the ESI-MS analysis (negative): m/z 221 [M-H]-; 1H
NMR (600 MHz, CD2Cl2) δ 9.61 (d, J = 7.0 Hz, 1 H), 9.51 (m, 2 H),
7.45 (s, 2 H), 5.82 (s, 2 H), 4.23 (s, 6 H).
(Tianjin,
China).
N-hydroxysuccinimide
(NHS),
1-(3-
dimethylaminopropyl)ꢀ 3-ethylcarbodiimide hydrochloride (EDC), and
3,3′,5,5′-tetramethylbenzidine (TMB) were obtained from Aladdin
Chemical Technology Co., Ltd. (Shanghai, China). Lactoferrin (LF) was
obtained from FUJIFILM Wako Pure Chemical Co. Ltd. (Japan). Bovine
serum albumin (BSA), keyhole limpet hemocyanin (KLH), complete and
incomplete Freund’s adjuvants, and polyethylene glycol solution
(Hybri-Max™) were obtained from Sigma-Aldrich (Shanghai, China).
Protein G resin and a secondary antibody (goat anti-mouse IgG, HRP
conjugated) were obtained from TransGen Biotech Co. Ltd. (Beijing,
China). Ninety-six-well polystyrene microplates were obtained from
Shenzhen Jincanhua Industry Co. Ltd. (Shenzhen, China). N,N-
dimethylformamide (DMF), dichloromethane (DCM), triethylamine
(TEA), Tween-20, methanol (MeOH), 1,4-dioxane, and petroleum ether
were obtained from Damao Chemical Reagent Co., Ltd. (Tianjin, China).
Gold nanoparticles (GNPs) were obtained from Wanlian Biotechnologies
Co. Ltd. (Guangzhou, China).
2.3.3. Hapten-C, 4-((2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)oxy)
butanoic acid
The same synthesis procedure was used for hapten-C as for hapten-B,
except ethyl bromoacetate was replaced by ethyl 4-bromobutyrate. The
following results were obtained from the ESI-MS analysis (negative): m/
z 249 [M-H]-; 1H NMR (600 MHz, CD2Cl2) δ 6.80 (dd, J = 6.1, 2.5 Hz,
1 H), 6.76 (dd, J = 9.0, 5.2 Hz, 2 H), 4.08 (t, J = 6.2 Hz, 2 H), 3.04 (s,
2 H), 2.60 (t, J = 7.3 Hz, 2 H), 2.12 (dd, J = 13.6, 6.6 Hz, 2 H), 1.49
(s, 6 H).
BALB/c female mice were purchased from the Guangdong Medical
Experimental Animal Centre and raised at the Animal Experiment
Centre of South China Agriculture University (Animal Experiment
Ethical Approval Number: 2019054, Fig. S1).
2.3.4. Hapten-D, 6-(((2-isopropylphenoxy)carbonyl)amino)hexanoic acid
The same synthesis procedure was used for hapten-D as for hapten-A,
except benzofuranol was replaced by 2-isopropylphenol. The following
results were obtained from the ESI-MS analysis (negative): m/z 292 [M-
H]-; 1H NMR (600 MHz, MeOD) δ 7.32 (dd, J = 5.8, 3.5 Hz, 1 H), 7.19
(dt, J = 4.6, 3.7 Hz, 2 H), 7.00 (dd, J = 5.9, 3.4 Hz, 1 H), 3.20 (t,
J = 6.9 Hz, 2 H), 3.13 (dt, J = 13.8, 6.9 Hz, 1 H), 2.33 (t, J = 7.4 Hz,
1 H), 1.67 (m, 1 H), 1.60 (m, 1 H), 1.44 (m, 1 H), 1.23 (t, J = 8.5 Hz,
3 H).
2.2. Instruments
A NanoDrop2000c spectrophotometer (Thermo Fisher, Shanghai,
China) was used to perform a UV–vis spectral scan to measure the
protein concentration. The plates used in the study were washed using a
Wellwasch™ microplate washer (Thermo Fisher, Hudson, NH, USA).
Other equipment included a MK3 microplate reader (Thermo Fisher,
Shanghai, China) and a DY6510 membrane strip reader (Wanlian Bio-
technologies Co. Ltd., Guangzhou, China).
2.3.5. Hapten-E, 2-(2-isopropylphenoxy)acetic acid
2.3. Hapten synthesis
The same synthesis procedure was used for hapten-E as for hapten-B,
except benzofuranol was replaced by 2-isopropylphenol. The following
results were obtained from the ESI-MS analysis (negative): m/z 193 [M-
H]-; 1H NMR (600 MHz, DMSO) δ 7.20 (dd, J = 7.5, 1.5 Hz, 1 H), 7.11
(m, 1 H), 6.91 (t, J = 7.4 Hz, 1 H), 6.81 (d, J = 8.2 Hz, 1 H), 4.68 (s,
2 H), 3.32 (dt, J = 13.9, 7.0 Hz, 1 H), 1.18 (d, J = 6.9 Hz, 6 H).
The uniform synthetic routes for the carbamate haptens are shown in
Fig. 1. The mass and NMR spectrograms used for sample characteriza-
tion are shown in the Supporting Material (Figs. S2–S9).
2.3.1. Hapten-A, 6-((((2,2-dimethyl-2,3-dihydrobenzofuran-7-yl)oxy)
carbonyl)amino) hexanoic acid
2.3.6. Hapten-F, 4-(2-isopropylphenoxy)butanoic acid
Benzofuranol (3.2 g, 20 mmol) was dissolved in 15 mL of DCM with
5 mL of TEA and stirred in an ice bath at 0 ◦C. Five milliliters of DCM
containing 4 g (20 mmol) of 4-nitrophenyl chloroformate were added
dropwise to the benzofuranol solution, and the reaction was allowed to
proceed overnight under stirring at room temperature. After the reaction
was completed, DCM was removed by evaporation; TEA was then
removed by dissolving the residue in ethyl acetate, followed by washing
three times with 0.5 M HCl. The organic phase was evaporated to
remove ethyl acetate. The residue was dissolved in 5 mL of 1,4-dioxane
and then added to a 6-aminocaproic acid solution (5 g, dissolved in
15 mL of saturated sodium carbonate solution), and the mixture was
maintained under stirring overnight. The crude intermediate product
was filtered, and HCl was added to adjust the pH of the mixture to 3–4.
The mixture was evaporated to remove water and purified. The
following results were obtained from the ESI-MS analysis (negative): m/
z 320 [M-H]-; 1H NMR (600 MHz, DMSO) δ 12.02 (s, 1 H), 7.68 (t,
The same synthesis procedure was used for hapten-F as for hapten-C,
except that benzofuranol was replaced by 2-isopropylphenol. The
following results were obtained from the ESI-MS analysis (negative): m/
z 221 [M-H]-; 1H NMR (600 MHz, DMSO) δ 12.13 (s, 1 H), 7.14 (m, 2 H),
6.88 (dd, J = 14.2, 7.6 Hz, 2 H), 3.97 (t, J = 6.2 Hz, 2 H), 3.24 (dt,
J = 13.8, 6.9 Hz, 1 H), 2.42 (t, J = 7.3 Hz, 2 H), 1.97 (p, J = 6.5 Hz,
2 H), 1.15 (dd, J = 8.8, 4.5 Hz, 6 H).
2.3.7. Hapten-G, 6-(((naphthalen-1-yloxy)carbonyl)amino)hexanoic acid
The same synthesis procedure was used for hapten-H as for hapten-A,
except benzofuranol was replaced by 1-naphthol. The following results
were obtained from the ESI-MS analysis (negative): m/z 300 [M-H]-; 1H
NMR (600 MHz, acetone) δ 10.47 (s, 1 H), 8.01 (dd, J = 5.6, 4.1 Hz,
1 H), 7.94 (m, 1 H), 7.77 (d, J = 8.3 Hz, 1 H), 7.54 (m, 2 H), 7.49 (t,
J = 7.9 Hz, 1 H), 7.32 (m, 1 H), 7.04 (dd, J = 10.8, 3.8 Hz, 1 H), 3.30
3

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Fig. 2. Surface electrostatic potential image of carbamate pesticide haptens. The red arrow highlights the strong negative charge of carbofuran and the corre-
sponding hapten; The yellow and blue arrow highlight the strong negative charge of amide of isoprocarb and carbaryl their corresponding hapten. (For interpretation
of the references to color in this figure legend, the reader is referred to the web version of this article.)
(dd, J = 13.0, 6.8 Hz, 2 H), 2.34 (t, J = 7.4 Hz, 2 H), 1.68 (m, 4 H),
except benzofuranol was replaced by 1-naphthol. The following results
were obtained from the ESI-MS analysis (negative): m/z 229 [M-H]-; 1H
NMR (600 MHz, MeOD) δ 8.23 (m, 1 H), 7.79 (m, 1 H), 7.43 (m, 4 H),
6.88 (d, J = 7.5 Hz, 1 H), 4.20 (t, J = 6.1 Hz, 2 H), 2.61 (t,
J = 7.3 Hz, 2 H), 2.23 (m, 2 H).
1.50 (m, 2 H).
2.3.8. Hapten-H, 1-naphthoxyacetic acid, CSA 2976-75-2
Hapten-H was obtained from Heowns Biochemical Technology Co.,
Ltd. (Tianjin, China).
2.4. Syntheses of immunogens and coating antigens
2.3.9. Hapten-I, 4-(naphthalen-1-yloxy)butanoic acid
The same synthesis procedure was used for hapten-F as for hapten-C,
The immunogen and coating antigen syntheses was performed
4