Monitoring of the organophosphate pesticide chlorpyrifos in vegetable samples from local markets in Northern Thailand by developed immunoassay

Article abstract of DOI:10.3390/ijerph17134723

Chlorpyrifos is an organophosphate pesticide that is wildly used among farmers for crop protection. However, there are concerns regarding its contamination in the environment and food chain. In the present study, an in-house indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) specific for detecting chlorpyrifos is developed and validated against gas chromatography–flame photometric detection (GC-FPD) as the conventional method. The developed ic-ELISA was used for detecting chlorpyrifos residue in vegetable samples. The developed ic-ELISA showed good sensitivity to chlorpyrifos at an IC₅₀ of 0.80 μg/kg, with low cross-reactivity to other organophosphate pesticides. The 160 samples were collected from local markets located in the Chiang Rai, Chiang Mai, and Nan provinces in northern Thailand. The positive rate of chlorpyrifos residues in the vegetable samples was 33.8percent, with the highest levels found in cucumbers, coriander, and morning glory, at 275, 145, and 35.3 μg/kg, respectively. The highest median levels of chlorpyrifos found in the detected samples were Chinese cabbage (332 μg/kg), cucumber (146.3 μg/kg) and Chinese Kale (26.95 μg/kg). The developed ic-ELISA is suitable for the rapid quantitation of chlorpyrifos residues.

Full text of DOI:10.3390/ijerph17134723

International Journal of
Environmental Research
and Public Health
Article
Monitoring of the Organophosphate Pesticide
Chlorpyrifos in Vegetable Samples from Local
Markets in Northern Thailand by
Developed Immunoassay
1
,
1
2
3
4
Surat Hongsibsong *, Tippawan Prapamontol , Ting Xu , Bruce D. Hammock , Hong Wang ,
Zi-Jian Chen 4 and Zhen-Lin Xu 4
1
Research Institute for Health Science, Chiang Mai University, Chiang Mai 50200, Thailand;
tippawan.prapamontol@cmu.ac.th
Beijing Key Laboratory of Biodiversity and Organic Farming, College of Resources and Environmental
2
Sciences, China Agricultural University, Beijing 100193, China; xuting@cau.edu.cn
Department of Entomology and Nematology and UCD Comprehensive Cancer Center, University of
3
California Davis, Davis, CA 95616, USA; bdhammock@ucdavis.edu
Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China
4
Agricultural University, Guangzhou 510642, China; gzwhongd@163.com (H.W.);
czj1q2w3e4r5t@163.com (Z.-J.C.); jallent@163.com (Z.-L.X.)
*
Correspondence: surat.hongsibsong@cmu.ac.th; Tel.: +66-5394-2508
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 16 May 2020; Accepted: 25 June 2020; Published: 30 June 2020
Abstract: Chlorpyrifos is an organophosphate pesticide that is wildly used among farmers for crop
protection. However, there are concerns regarding its contamination in the environment and food chain.
In the present study, an in-house indirect competitive enzyme-linked immunosorbent assay (ic-ELISA)
specific for detecting chlorpyrifos is developed and validated against gas chromatography–flame
photometric detection (GC-FPD) as the conventional method. The developed ic-ELISA was used
for detecting chlorpyrifos residue in vegetable samples. The developed ic-ELISA showed good
sensitivity to chlorpyrifos at an IC50 of 0.80 µg/kg, with low cross-reactivity to other organophosphate
pesticides. The 160 samples were collected from local markets located in the Chiang Rai, Chiang Mai,
and Nan provinces in northern Thailand. The positive rate of chlorpyrifos residues in the vegetable
samples was 33.8%, with the highest levels found in cucumbers, coriander, and morning glory, at 275,
145, and 35.3 µg/kg, respectively. The highest median levels of chlorpyrifos found in the detected
samples were Chinese cabbage (332 µg/kg), cucumber (146.3 µg/kg) and Chinese Kale (26.95 µg/kg).
The developed ic-ELISA is suitable for the rapid quantitation of chlorpyrifos residues.
Keywords: chlorpyrifos residue; ic-ELISA; GC-FPD; northern Thailand
1
. Introduction
Chlorpyrifos (O,O-diethyl O-(3,5,6-trichloro-2-pyridinyl) phosphorothioate) is a broad-spectrum
organophosphate pesticide, which is the most popular among farmers for protecting their crops by
controlling different kinds of pests [ ]. It is extremely toxic to a wide range of nontarget aquatic
organisms [ ]. Additionally, the consumer’s health risk is a concern due to presence of residues in
fruits and vegetables [ ], the environment [ ], and all of the agricultural products [ ]. The is
1
2
3,
4
5,
6
7–9
possible because farmers use high volumes of chlorpyrifos, and thus it has a chance to be detected
in many samples and agricultural production in general. Chlorpyrifos can inhibit cholinesterase,
i.e., acetyl cholinesterase and butyryl cholinesterase [10
,11], and can be cause damage to the central
nervous system [12], as well as damage to the sympathetic and parasympathetic nervous systems [13].
Int. J. Environ. Res. Public Health 2020, 17, 4723; doi:10.3390/ijerph17134723
www.mdpi.com/journal/ijerph

Int. J. Environ. Res. Public Health 2020, 17, 4723
2 of 14
Moreover, in human epidemiological studies, occupational exposure to chlorpyrifos has been related to
neurological and neuro-behavioral deficits, including cognitive impairment [14 18]. Chlorpyrifos also
–
has been shown to produce learning deficits in rats after acute and repeated administration, similar to
those induced in Alzheimer’s disease (AD) [19]. Therefore, chlorpyrifos has been listed as banned
in several countries, including Thailand. However, monitoring still needs to be carried out given its
illegal use, because it is a very toxic substance.
Currently, most of the techniques and standard methods for detecting chlorpyrifos are
chromatographic based-methods, i.e., liquid chromatography [20], gas chromatography [
8,21,22],
and mass spectrometric methods [23 25]. Chromatographic-based methods have complicated steps,
–
require skilled technical help, are equipment intensive, require a skilled person, and require a significant
amount of time. Therefore, a low number of samples can be analyzed per day, and thus a high number
of samples cannot be processed in a short time. Additionally, it also very expensive per sample.
Thus, the immunoassay complements instrumental methods in many ways, in particular being more
applicable in handling a higher number of samples and field assays [26–30]. The absorbance or color are
inversely proportional to compounds in the competitive immunoassay. It is useful to use immunoassay
as a tool for detecting chlorpyrifos in a high number of samples for epidemiology studies.
This paper reports on a developed immunoassay for detecting the organophosphate pesticide
chlorpyrifos based on mouse polyclonal antibody and its application for the monitoring of chlorpyrifos
residues in vegetable samples from the Chiang Mai, Chiang Rai, and Nan provinces in the northern
part of Thailand. Analytical parameters, such as the sensitivity, accuracy, and reproducibility, were
evaluated, and the results are discussed in comparison with GC-FPD. The residues in vegetable samples
were analyzed by using the developed immunoassay as an analytical method.
2
. Materials and Methods
2
.1. Chemicals and Reagents
The 3-mercaptopropionic acid, potassium hydroxide (KOH), potassium iodide (KI) hydrochloric
acid (HCl), sodium hydroxide (NaOH), bovine serum albumin (BSA), and ovalbumin (OVA) were
obtained from Sigma-Aldrich Chemie (Steinheim, Germany). The Bio-Rad protein assay kit was from,
BioRad (California, USA). Sodium chloride, tetrabutylammonium iodide (TBAI), disodium hydrogen
phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, potassium chloride,
sodium carbonate, and citric acid were from Merck (Hohenbrunn, Germany). The organic solvents,
i.e., acetonitrile, dimethylformamide, methanol (MeOH), and ethyl acetate, were from J.T. Baker
0
0
(
Pennsylvania, USA). The 3,3 ,5,5 -tetramethylbenzidine (TMB) was from Fluka (Steinheim, Germany),
and the tetrahydrofuran (THF), methanol (MeOH) dimethyl formamide (DMF), and dimethyl sulfoxide
DMSO) was from Sigma-Aldrich (Steinheim, Germany). Goat anti-mouse IgG (H+L) was from
Invitrogen (Rockford, USA).
(
2
.2. Standard Organophosphate Pesticides
Eighteen organophosphate pesticide (OPs) standards (purity 93–99%) were purchased from Dr.
Ehrenstorfer (Augsburg, Germany) and used for cross-reactivity analysis by immunoassay and analysis
by gas chromatography–flame photometric detection (GC-FPD), i.e., chlorpyrifos-methyl (purity 98.7%),
dichlorvos (purity 98.7%), mevinphos (purity 96.9%), omethoate (purity 93.4%), dicrotophos
(
purity 97.5%), monocrotophos (purity 98%), dimethoate (purity 99.3%), diazinon (purity 98.9%),
parathion-methyl (purity 98.2%), fenitrothion (purity 98.6%), malathion (purity 99.4%), chlorpyrifos
purity 99%), primiphos-ethyl (purity 99%), methidathion (purity 98.3%), prothiophos (purity 95.4%),
(
profenofos (purity 99%), ethion (purity 98.8%), triazophos (purity 98.9%), ethyl4-nitrophenyl
phenylphosphonothioate (EPN) (purity 98.2%), azinphos-ethyl (purity 99.4%), and azinphos-methyl
(
purity 99.6%). The standard pesticides were prepared in methanol and diluted in 20% methanol in
◦
PBS (pH 7.2) before being analyzed by ic-ELISA, and was also stored at −20 C prior to the analysis.

Int. J. Environ. Res. Public Health 2020, 17, 4723
3 of 14
2
.3. Development of Immunoassay for Detecting Chlorpyrifos
The immunoassay was developed in accordance to a similar protocol as in previous studies,
such as a hapten synthesis, preparation of immunogens and coating antigens, characterizing the
polyclonal antibody, and the matrix effect of the sample extract on the antibody [31–34].
2
.3.1. Hapten Synthesis
The hapten was synthesized by following a previously published method [35,36], with some
modification. The haptens were used for preparation of the immunogens and coating antigens as well
as the developed immunoassay. The haptens were synthesized as follows in Figure 1:
Figure 1. The synthesis of haptens was performed as previously described [32]. Compounds (
and (c) were separated by column chromatography.
a), (b),
The structures of the haptens are shown in Figure 1. Synthetic intermediates were analyzed by
1
13
thin layer chromatography (TLC) and confirmed by H- and C-nuclear magnetic resonance (NMR).
(
1) Hapten (a) and (b): To a solution of chlorpyrifos (1.00 g, 2.85 mmol) dissolved in ethanol (5 mL),
KOH (400 mg, 7.13 mmol) was added and stirred for 10 min. The resulting solution was treated
◦
with 3-mercaptopropanoic acid (0.30 mL, 3.45 mmol), and the temperature was raised to 80 C
and refluxed for 1 h. The solvent was removed under reduced pressure, followed by washing
three times with hexane (20 mL each time). The residue was acidified to a pH of 2 and extracted
with 20 mL of dichloromethane three times. The organic phase was dried over sodium sulfate
and concentrated under reduced pressure to give the crude product, which was purified by silica
gel column chromatography. The product was mixed between (a) and (b) in a 2:1 ratio with
1
an 83% yield (0.68 g). Hapten (a), H NMR (CDCl , 400 MHz)
(
δ
7.65 (s, 1H), 4.33 (m, 4H), 3.41
3
13
t, J = 7.2 Hz, 2H), 2.90 (t, J = 7.2 Hz, 2H), 1.41 (t, J = 7.2 Hz, 6H); C NMR (CDCl , 100 MHz)
176.6, 153.6, 151.4, 138.9, 125.0, 116.0, 66.4, 66.3, 34.1, 25.3, 15.9, 15.8 and hapten (b), H NMR
7.65 (s, 1H), 4.33 (m, 4H), 3.41 (t, J = 7.2 Hz, 2H), 2.90 (t, J = 7.2 Hz, 2H), 1.41
3
1
δ
(
(
CDCl , 400 MHz)
δ
3
13
t, J = 7.2 Hz, 6H); C NMR (CDCl , 100 MHz)
δ 176.6, 153.6, 151.4, 138.9, 125.0, 116.0, 66.4, 66.3,
3
3
4.1, 25.3, 15.9, 15.8.
2) Hapten (c): To a solution of chlorpyrifos (1.00 g, 2.85 mmol) dissolved in 3:2:1 THF/MeOH/water
5 mL), LiOH (82 mg, 3.42 mmol) was added and stirred for 30 min. The resulting solution
was evaporated before adding DMF and then treated with methyl-6-bromohexanoate (715 mg,
.42 mmol) and a catalytic amount of TBAI and KI. After refluxing for 1 h, the reaction mixture was
allowed to cool down to room temperature before further hydrolysis with NaOH. After completion
(
(
3

Int. J. Environ. Res. Public Health 2020, 17, 4723
4 of 14
of the reaction, the residue was acidified to a pH of 2 and extracted with 20 mL of dichloromethane
three times. The extraction phase was dried over sodium sulfate and concentrated under reduced
pressure to give the crude product, which was purified by silica gel column chromatography.
1
The product was obtained with a 69% yield (0.62 g). H NMR (CDCl )
δ
7.74 (s, 1H, ArH), 4.92
3
(
s, 2H, OCH2); 13C NMR (CDCl , 100 MHz) δ 171.0, 158.6, 145.7, 140.2, 127.2, 120.7, 65.6.
3
2
.3.2. Preparation of the Immunogen and Coating Antigen
The mixed carboxylic acid hapten was attached to both a protein and an enzyme using the active
ester method [37]. Hapten was conjugated to bovine serum albumin (BSA) for immunogen preparation
and conjugated to oval albumin (OVA) for coating antigen preparation. The hapten densities of the
immunogen and coating antigen were estimated based on mass spectra of ultraviolet spectra to confirm
coupling and then calculated [38]. The synthesized immunogen and coating antigen demonstrated
qualitative differences between the corresponding carrier protein and conjugate in the UV–Vis spectra,
indicating that the hapten had been conjugated to the carrier protein successfully. The estimation
of molar absorptivity of the hapten was the same for the free and conjugated forms of the carrier
protein. The estimated molar ratio of the immunogen and coating antigen were 12 and 22, respectively.
The protein contents of the hapten–protein conjugates in the dialysates were determined according to
the Bio-Rad dye based on the Bradford protein assay.
2
.3.3. Preparation of Polyclonal Antibody (pAb)
The animals were reared in a clean, standard environment, with a food and water supply.
The immunization experimental protocol with animals was performed in accordance with relevant
institutional and national guidelines and regulations, and it was approved by the Animal Care and Use
Committee, Chiang Mai University (Protocol number: 2561/MC-004). Two BALB/c mice (age 6–8 weeks)
were immunized so their immune system would produce polyclonal antibodies by using the prepared
immunogen for induction of antibody responses against hapten according to previous studies [38
Two mice were inoculated with 100 g of immunogen dissolved in 10 mM phosphate buffer saline (PBS)
and then emulsified with complete Freund’s adjuvant (1:1, v/v) injected through the subcutaneous (s.c.)
route at multiple sites on the back. Then, they were given three booster immunizations with 100 g of
,39].
µ
µ
immunogen dissolved in PBS and then emulsified with incomplete Freud’s adjuvant (1:1, v/v) with
substitution with Freund’s complete adjuvant every two weeks in the same way. Mice were bled from
the tail veins before the immunization schedule first and then every week. The serum was separated
from the blood by centrifugation and stored in the freezer prior to use for evaluation of the antibody
response against hapten by direct ELISA using hapten-OVA as the coating antigen. The serum from
a sensitive mouse from the 3rd to 5th immunization were collected from the tail (0.3 mL each time).
After the 6th immunization for 3 days, the heart blood of the mice was collected. Mice were placed in a
deep sleep by saturated carbon dioxide before heart blood collection. All the collected serums were
mixed and the pooled serum from the heart blood was prepared (the reciprocal titer about 51,200) and
used for further experiments.
2
.3.4. Development of ic-ELISA
1) Methanol effect: Methanol is a good solvent for immunoassays and has been used in many previous
studies [40 42]. However, the methanol content in PBS may affect the antibody. The methanol
contents in PBS were studied by using different concentrations of methanol, i.e., 50%, 40%, 20%,
0%, and 5%, in PBS as a diluent for chlorpyrifos in several concentrations. The absorbance of
(
–
1
each methanol content and IC50 were compared, and due to the good results and no effect from
methanol, it was selected as the diluent of the developed immunoassay.
(
2) Ionic strength: The ionic strength affected the ic-ELISA, and thus the standard curves of chlorpyrifos
were analyzed by using different concentrations of 10 mM PBS at a pH of 7.0, i.e., 1x, 2x, 3x, 4x,
5
x, and DI water.

Int. J. Environ. Res. Public Health 2020, 17, 4723
5 of 14
(
3) Indirect Competitive ELISA (ic-ELISA): The ic-ELISA was performed according to the method of
Hongsibsong et al. [39]. The concentrations of antibody and coating antigen were optimized
by checkerboard titration. The good condition was coating the antigen at 1 g/mL and a serum
dilution at 1:1000. The ic-ELISA was performed by using the optimal concentration as follows.
Microtiter plates (Maxisorb, NUNC, Roskilde, Denmark) were coated with 100 L/well of the
hapten-OVA (1 g/mL) as a coating antigen in a carbonate buffer at a pH of 9.6 and allowed
to sit overnight at 4 C. The plates were washed with PBS plus 0.05% Tween 20 (PBST) and
blocked with 200 L/well of 1% (w/v) gelatin in PBS at a pH of 7.2. After 1 h of incubation at room
µ
µ
µ
◦
µ
temperature, the plates were washed as described previously. Standards (or samples extracted)
were mixed with equal volumes of serum diluted in PBS (1:1000) and pre-incubated for 1 h at
room temperature. The pre-incubated mixture was transferred to the wells (100
incubated for 1 h at room temperature for competition. Then, the plate was washed by PBST,
and 100 L/well of 1:5000 HRP conjugated goat anti-mouse IgG (H+L) in PBS at a pH of 7.2
was added. After 1 h, the plate was washed, and 100 L of a substrate solution (0.1 mL of 1%
H O and 0.4 mL of 0.6% 3,3 ,5,5 -tetramethylbenzidine in dimethyl sulfoxide (DMSO) were
µL/well) and
µ
µ
0
0
2
2
added to 25 mL of citrate-acetate buffer, pH = 9.6) was added to each well. The plates were
stopped with 50 L of 2N H SO and read by an ELISA plate reader (Sunrise, Salzburg, Austria)
µ
2
4
at 450 nm. The development of a yellow color was inversely proportional to the amount of
chlorpyrifos present. The absorbance was calculated for 50% inhibition by a nonlinear curve fit.
The concentration of chlorpyrifos residue was calculated from the standard curve.
(
4) Cross-reactivity (CR): The cross-reactivity was studied by ic-ELISA and substitution of the
chlorpyrifos standard or sample with an organophosphate pesticide in the same group
as chlorpyrifos. Organophosphate pesticide standards were used for cross-reactivity by
immunoassay, i.e., chlorpyrifos-methyl, dichlorvos, mevinphos, omethoate, dicrotophos,
monocrotophos, dimethoate, diazinon, parathion-methyl, fenitrothion, malathion, chlorpyrifos,
primiphos-ethyl, methidathion, prothiophos, profenofos, ethion, triazophos, ethyl 4-nitrophenyl
phenylphosphonothioate (EPN), azinphos-ethyl, and azinphos-methyl. The cross-reactivity was
determined according to the equation below:
CR (%) = (IC50 (chlorpyrifos) / IC50 (interferent)) × 100.
(1)
(
5) Matrix effect of the color from the vegetable sample: Since vegetables have colors, the effects of
the various colors of vegetables on the antibody were studied. The green (kale), red (tomato),
and white (Chinese cabbage) colors, which are commonly consumed in the Thai community,
were evaluated. The vegetable samples were chopped into small pieces and extracted following
a previously described method [4,43]. The best methanol content was used and the effect of
the extraction of each colored vegetable on the antibody was determined. The recovery was
computed by spiking chlorpyrifos into a pooled vegetable sample and then extracting before
analyzing. The pooled vegetable was prepared from those three kinds of vegetables with no
chlorpyrifos residues after analyzing by GC-FPD.
(
6) Immunoassay validation: To evaluate the performance of the developed ic-ELISA for
organophosphate pesticide chlorpyrifos, three experiments were performed: (1) the recoveries of
spiked pooled vegetable samples were measured by ic-ELISA, and the accuracy, precision, limit
of detection (LoD), and limit of quantification (LoQ) were reported as the percent of recovery,
percent of coefficient of variance (%CV), IC , and IC , respectively; (2) the applicability of
15
20
ic-ELISA to detect chlorpyrifos was determined by analyzing 70 blind vegetable samples, and the
results were compared against the results from GC-FPD, which is the standard technique with a
specific detector for analyzing compounds that include phosphate in their molecule; and (3) the
application of the developed ic-ELISA to analyze the organophosphate pesticide chlorpyrifos
in vegetable samples. The vegetable samples, i.e., coriander (n = 28), yard long bean (n = 23),
cabbage (n = 17), pakchoi (n = 17), Chinese cabbage (n = 13), morning glory (n = 13), cauliflower

Int. J. Environ. Res. Public Health 2020, 17, 4723
6 of 14
(n = 9), spring onion (n = 8), broccoli (n = 7), chili (n = 7), eggplant (n = 6), Chinese kale (n = 5),
cucumber (n = 4), and tomato (n = 3), were collected from local markets in the Maetang district,
Chiang Mai province (n = 28); Phaya Mengrai district, Chiang Rai province (n = 77); and Muang
district, Nan province (n = 55)—for a total of 160 samples. Five kilograms of each sample was
collected in each market; the edible parts were finely chopped and 300 g was randomly taken for
analysis. Then, all of the samples were transferred to the Toxicology Laboratory, Environmental
and Health Research Unit, Research Institute for Health Science, Chiang Mai University, and kept
◦
in a −20 C freezer prior to analysis.
2
.3.5. Analysis of the Organophosphate Pesticide Residues in the Vegetable Samples
(
1) Sample extraction and clean up: The sample extraction followed the method of [4,39]. Briefly, 5 g of
vegetable sample was weighed into a 50 mL centrifuge tube, followed by the addition of 10 mL of
acetonitrile (high-performance liquid chromatography grade). Two hundred fifty microliters of
5
µg/mL triphenylphosphate (internal standard (IS)) was added and subsequently centrifuged
for 5 min at 2500 rpm. The supernatant was transferred to a 50 mL centrifuge tube with the
addition of 6 g of MgSO and 3 g of NaCl, followed by centrifugation again for 5 min at 2500 rpm.
4
The extract was evaporated to complete dryness using a vacuum rotary evaporator (Buchi, Flawil,
◦
Switzerland) with a water bath at 30 to 35 C and then reconstituted with 5 mL of ethyl acetate.
One milliliter of ethyl acetate phase was pipetted into 2 dispersive solid-phase extraction tubes
and centrifuged for 3 min at 2000 rpm. Finally, the extract was evaporated with a gentle stream
of nitrogen at room temperature and subsequently reconstituted in 1.0 mL of 10% methanol in
PBS at a pH of 7.0 for the immunoassay, and another one was reconstituted with 0.5 mL of ethyl
acetate for gas chromatography (GC) analysis.
(
2) Gas chromatography–flame photometric detection (GC-FPD): A Hewlett-Packard model 6890 equipped
with a flame photometric detector, a capillary column (DB-5MS, 0.25 mm
×
I.D.
×
30 m length
×
0.25 m film thickness (Agilent J & W column; Agilent Technologies, DE, USA), and a
µ
computerized data handling system (GC Chemstation A.10.02; Agilent Technologies, CA, USA)
◦
was used. The temperature was 220 C for the injection port (splitless mode). The temperature
programming of the oven was as follows: initial temperature of 100 C for 10 min, first ramp at
◦
◦
◦
◦
◦
1
5 C/min to 180 C (5 min), second ramp at 5 C/min to 250 C (3 min), and the final temperature
◦
maintained at 290 C for 4 min. The carrier gas was 99.999% helium.
3
. Results and Discussion
3
.1. Development of the Immunoassay
Since chlorpyrifos (chlorpyrifos-ethyl) has been imported to Thailand, and in a previous study
shown to be the most detected residue in vegetable samples [4], a simpler method was required for
detecting chlorpyrifos in a high number of samples. The study developed an in-house immunoassay
and applied it for detecting chlorpyrifos in vegetable samples. The hapten was synthesized by following
methods previously published [35
,36]. It was confirmed that the procedure for preparing the hapten
could be based on a previous study [35
,36], which published a structure of hapten (hapten (a)) that
could be used to produce the antibody to chlorpyrifos. The coating antigen prepared by using the same
hapten with immunogen did not give good sensitivity for ic-ELISA (data not shown). The polyclonal
antibody in the serum from a mouse was used for developing the immunoassay by using the pooled
serum from after the 3rd immunization of heart blood (3.0 mL) because the titer of antibody in the
serum samples was equal and used for analyzing the vegetable samples. The dilution of serum at
1
:1000 was used for the determination of chlorpyrifos and the validation of the ic-ELISA, along with a
good coating antigen, namely, (c)-OVA (compound (c) in Figure 1), which gave the lowest concentration
inhibition of chlorpyrifos at an IC50 of 0.80 µg/kg.

Int. J. Environ. Res. Public Health 2020, 17, 4723
7 of 14
Sensitivity and specificity based on the optimized ic-ELISA and the standard curves of
chlorpyrifos were developed. The developed ic-ELISA showed high sensitivity to chlorpyrifos
with an IC50 = 0.80
±
0.56 µg/kg. The cross-reactivities (CRs) with dichlorvos, mevinphos, omethoate,
dicrotophos, monocrotophos, dimethoate, diazinon, parathion-methyl, fenitrothion, malathion,
primiphos-ethyl, prothiophos, ethion, triazophos, ethyl 4-nitrophenyl phenylphosphonothioate (EPN),
azinphos-ethyl, and azinphos-methyl at very high concentrations were minimal (>1000
The pAb gave a cross-reactivity with chlorpyrifos-methyl, profenofos, and methidathion at 125%,
4.28%, and 0.28%, respectively. Chlorpyrifos can be determined at concentrations ranging from 20.0
to 2090 g/kg. The IC50 of the present study had both higher [44 45] and lower [45 46] values than
µg/kg).
1
µ
,
,
previously reported. The specificity of the produced pAb was good for detecting chlorpyrifos in the
sample because it had low cross-reactivity to other OPs, except for chlorpyrifos-methyl, which has
no reported use in Thailand (the Office of Agricultural Regulation), and pAb can bind the group of
organophosphate pesticides in a smaller number with a high concentration. The hapten with a similar
structure to that in the present study gave the same results [45], and this was helpful for developing
methods for detecting chlorpyrifos in real samples.
The matrix effect is one of the main factors that restricts the application of immunoassays
in pesticide analysis [32
samples, the direct extraction of pesticides from food samples with organic solvents, such as methanol,
is commonly used [40 42]. In practice, a dilution of 20–100 times of the methanol extraction should be
,33,47]. In the development of immunoassays for pesticides in vegetables
–
done to reduce the matrix effect prior to immunoassays. However, the dilution can cause a reduction
in the assay sensitivity, and an overlarge dilution would cause inapplicability of the immunoassays.
To confirm the applicability based on the developed ic-ELISA, different colored vegetable samples were
used as matrix substances. The samples of three colors (green from kale, white from Chinese cabbage,
and red from tomato) were used to determine the effect on the assay at different concentrations of 25, 50,
and 100 µg/kg compared with 10% methanol in PBS at a pH of 7.2. The results are shown in Table 1 and
confirmed that color had an effect on the binding, but it did not make a significant difference. As color
had an effect on the assay, pooled samples were prepared from those three kinds of vegetables with no
chlorpyrifos residue after analyzing by GC-FPD. It is good to have used vegetables of different colors,
but it does not imply the matrix effects are only due to color. The pooled samples were extracted and
used as a matrix for a standard curve for calculating the concentration of chlorpyrifos in the samples.
Due to the effect of the organic solvent and the matrix effect, the pooled samples were extracted by
using the methods as previously reported. Spiked pooled control samples were extracted between
every 20 unknown samples.
Table 1. Effects of the organic solvent, PBS, and matrix effect from the color of vegetables on ic-ELISA.
Methanol
IC50 (µg/kg)
PBS, pH = 7.2
IC50 (µg/kg)
5
2
1
0%
5%
0%
1.60 ± 0.60
0.80 ± 0.12
0.40 ± 0.24
0.52 ± 0.33
1×
2×
3×
4×
1.40 ± 0.68
1.20 ± 0.56
1.80 ± 0.62
2.20 ± 1.02
4.00 ± 2.24
10.0 ± 6.78
5
%
5
×
DI water
Matrix effect from the colors of vegetable samples
IC50 (µg/kg)
Tomato
Kale
Chinese cabbage
0.90 ± 0.42
0.90 ± 0.38
1.40 ±0.66

Int. J. Environ. Res. Public Health 2020, 17, 4723
8 of 14
3
.2. The Optimization of ic-ELISA for Detecting Chlorpyrifos
The optimization parameters are shown in Table 2. The recoveries of chlorpyrifos from the spiked
pooled vegetable samples ranged from 95.3% to 117.8% (mean = 102.9%). The precision was reported
by the coefficient of variation (CV), and the CV ranged from 4.6% to 6.7%, indicating that the developed
ic-ELISA can be used for the detection of chlorpyrifos with good reproducibility. The sensitivity was
evaluated by determining the limit of detection (LOD) and limit of quantification (LOQ) using the
IC20 to IC80 (0.40 to 41.8
and LOQ values were lower than the MRLs established by Codex (Codex Alimentarius Commission,
019) for vegetables samples. The ability of the developed ic-ELISA was determined: the positive
µg/kg). The LOD for the chlorpyrifos at the IC15 was 0.26 µg/kg. The LOD
2
sample was the detected sample by GC-FPD; the negative sample was not detected by GC-FPD; and
the positive samples by this method and GC-FPD were 46 and 33 samples, respectively. The results
2
show good correlation with the results from GC-FPD (R linear = 0.910), where a value of 0.954 was
obtained by the Pearson correlation test. The results from ic-ELISA provided overestimated qualitative
results, these maybe from the matrix effect and very high sensitivity of the ic-ELISA. The pretreatment
of samples could improve the sensitivity because of less dilution [47]. The developed ic-ELISA shows
good specificity to chlorpyrifos and chlorpyrifos-methyl, while the previous report showed broad
specificity to a group of pesticides, making it unable to quantify a single pesticide within the same
sample. The low cross-reactivity to other compounds in the group of OPs led to good specificity to
chlorpyrifos and good accuracy to identify the residue in real samples. To validate the result from
ic-ELISA by GC-FPD, there was thirteen false positive samples with no false negatives, so that could
be applied as both a screening test and quantitative test with a calibration curve. The present study
applied the developed ic-ELISA to quantify the single OP, i.e., chlorpyrifos, and the positive sample
was confirmed by GC-FPD. The high concentrations above the limit of quantification were diluted
and repeated. The concentrations of chlorpyrifos residues in vegetables were calculated against the
standard curve (Figure 2) and reported in
µg/kg of vegetable sample. The results of the immunoassay
were usually validated with the gold standard technique, i.e., a chromatographic technique [34
,
40,48].
Figure 2. Standard curve of organophosphate pesticide chlorpyrifos by optimized ic-ELISA. The coating
antigen was (c)-ova 1µg/ml, and the serum dilution was 1:1000.

Int. J. Environ. Res. Public Health 2020, 17, 4723
9 of 14
Table 2. Accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ) of the
developed ic-ELISA.
Parameters
ic-ELISA
0.26 (IC )
GC-FPD
LOD, µg/kg
LOQ, µg/kg
1.00
2.00
1
5
0.40–41.8 (IC –IC )
2
0
80
Precision (%CV, n = 4)
2
1
0
.00 µg/kg
.00 µg/kg
.50 µg/kg
4.60
6.70
5.50
2.00
2.58
3.40
Accuracy (%Recovery: n = 5)
2
1
0
.00 µg/kg
.00 µg/kg
.50 µg/kg
95.3
117.8
95.72
114.32
99.42
97.60
3
.3. Monitoring of Chlorpyrifos Residue in Vegetable Samples from Northern Thailand
Currently, there are already several studies that indicate that ELISA can be used to analyze
agricultural products and food samples after solvent extraction [40,49–51]. The developed ic-ELISA is
very useful for analyzing samples in a high number: it is a simple process, using inexpensive equipment,
and done in a rapid manner. The conventional methods, such as GC-FPD, used 50 min per sample
(one run), while ic-ELISA can analyze 50 samples in one day in approximately 4 h. The developed
ic-ELISA exhibited good accuracy and reproducibility, and it is ideally suited as a fast, high-throughput,
and low-cost screening test for organophosphate (OP) residues prior to chromatographic analysis.
The quality control of ic-ELISA was assessed by analyzing chlorpyrifos intra-batch (analyzing within
group of samples, n = 5) and inter-batch (analyzing between groups of samples, n = 10). The results
showed good intra-batch (%CV = 11.8) and inter-batch (%CV = 16.3), respectively. Table 3 reports
the concentration of chlorpyrifos in vegetables surveyed from local markets in three provinces of
northern Thailand in 2016, as well as comparison with EU-MRL for assessing the situation of residue
chlorpyrifos in vegetable samples from northern Thailand. The frequency that chlorpyrifos residues
was found in the vegetable samples was 33.8%, and the highest levels were found in cucumbers,
coriander, and morning glory, at 145, 38, and 33
chlorpyrifos was detected in all kinds of vegetable samples. The highest chlorpyrifos level was found
in cucumber (275 g/kg), followed by coriander (145 g/kg) and Pakchoi (60.6 g/kg). Previous studies
reported chlorpyrifos residues in coriander, Chinese cabbage, broccoli, and Chinese kale [ 52], as well
µg/kg, respectively. Among the compounds detected,
µ
µ
µ
4
,
as in mock pakchoi [53], with equal concentrations and detection of chlorpyrifos in the same types
of vegetables.
The findings showed that the median concentration of the detected chlorpyrifos for all the
vegetables was above the maximum residue limits established by the European Union, except for
Thai eggplant and broccoli. No Thai-MRL is yet established for chlorpyrifos residues in vegetables,
i.e., water convolvulus, mock pakchoi, and yard long bean. It is concluded that awareness, safety
education, and strict regulation of pesticide use is still necessary.

Int. J. Environ. Res. Public Health 2020, 17, 4723
10 of 14
Table 3. The chlorpyrifos residue in vegetable samples from northern Thailand.
Chlorpyrifos Residue
Maximum Residue Limit
Vegetables
Samples
Number of
Samples
(µg/kg)
Median
Min–Max
(µg /kg)
Thailand (1)
Codex (2)
EU (3)
Positive Number
Mean ± SD
Coriander
Yard Long Bean
Pakchoi
28
23
17
17
13
13
9
8
7
7
6
14
10
3
2
1
3
3
6
2
3
1
2
2
2
54
38.3 ± 38.4
16.6 ± 15.5
30.8 ± 27.8
13.4 ± 0.01
332 ± 0.00
32.8 ± 2.14
16.4 ± 9.05
18.6 ± 8.97
9.00 ± 0.42
24.5 ± 8.25
6.20 ± 0.00
30.0 ± 23.8
146 ± 183
24.05
12.35
26.30
13.45
332.3
31.7
13.00
16.45
9.00
23.0
6.20
26.95
146.3
23.50
18.40
3.60–145
3.20–56.0
5.60–60.6
9.00–17.9
332
31.5–35.3
9.60–26.7
6.10–29.6
8.70–9.30
17.10–33.4
6.20
-
-
-
-
-
-
-
-
-
-
-
10
10
10
10
10
10
50
10
10
10
10
10
10
10
-
Cabbage
100
100
-
Chinese Cabbage
Morning glory
Cauliflower
Spring onion
Broccoli
50.0
200
2,000
-
-
Chili
3000
Thai Eggplant
Chinese Kale
Cucumber
Tomato
200
100
-
5
4
3
160
10.1–43.8
16.9–275
18.2–28.8
3.2–332
-
-
-
-
-
23.5 ± 7.50
35.3 ± 58.4
2000
-
Total
Note: (1) Thai Food and Drug Administration (2017), (2) Codex Alimentarius Commission (2019), (3) European Commission (2020).

Int. J. Environ. Res. Public Health 2020, 17, 4723
11 of 14
4
. Conclusions
The present study shows the ic-ELISA developed from pAb exhibited reliable and high sensitivity
for chlorpyrifos detection. In comparison with other ELISA approaches, this developed ELISA exhibited
good resilience against organic solvents (10% methanol) and a large linear range that spans two orders
of magnitude. The developed ELISA was suitable for different levels of chlorpyrifos in samples.
The developed inhouse immunoassay method can be used for analyzing chlorpyrifos residue in a
high number of vegetable samples, and it is rapid, inexpensive per sample, and can be used for a
relevant diversity of samples. The data of chlorpyrifos residues in vegetable samples from the northern
part of Thailand detected in many kinds of samples contained chlorpyrifos. Chlorpyrifos residues
were the most frequently detected in coriander, with 10 samples (35.71%) exceeding the EU-MRL,
while spring onion had the highest percent of detection (75%) exceeding the EU-MRL. A positive
sample should be confirmed by standard analytical technique, such as GC-FPD. The contamination
levels of chlorpyrifos residues could be considered a possible public health problem because it is the
most popular insecticide used among farmers and one of the highest volumes of organophosphate
insecticides imported into Thailand.
Author Contributions: Conceptualization, S.H., T.P., T.X., H.W., B.D.H. and Z.-L.X.; methodology, S.H., B.D.H., T.P.;
validation, S.H., Z.-J.C., and Z.-L.X.; formal analysis, S.H. and Z.-J.C.; investigation, S.H., T.P., B.D.H., and Z.-L.X.;
resources, Z.-L.X. and T.X.; data curation, S.H.; writing—original draft preparation, S.H.; writing—review and
editing, S.H., T.P., and B.D.H.; supervision, S.H. and T.P.; project administration, S.H.; funding acquisition, S.H.
and T.P. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by The Thailand Research Fund (No. TRG6080007), Chiang Mai University,
and partial support came from NIEHS/Superfund Research Program P42 ES004699 Biomarkers of exposure to
hazardous substances.
Acknowledgments: The authors would like to thank The Thailand Research Fund (No. TRG6080007), Chiang
Mai University, and partial support came from NIEHS/Superfund Research Program P42 ES004699 Biomarkers of
exposure to hazardous substances. We also offer our thanks to Prof. Mookda Pattarawarapan and Assoc. Prof.
Wong Phakhodee, Department of Chemistry, Faculty of Science, Chiang Mai University for the preparation of
the haptens.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
Tawatsin, A.; Thavara, U.; Siriyasatien, P. Pesticides used in Thailand and toxic effects to human health.
Med. Res. Arch. 2015, 3, 1–10. [CrossRef]
Simon, D.; Helliwell, S.; Robards, K. Analytical chemistry of chlorpyrifos and diuron in aquatic ecosystems.
Anal. Chim. Acta 1998, 360, 1–16. [CrossRef]
Chen, C.; Qian, Y.; Liu, X.; Tao, C.; Liang, Y.; Li, Y. Risk assessment of chlorpyrifos on rice and cabbage in
China. Regul. Toxicol. Pharmacol. 2012, 62, 125–130. [CrossRef] [PubMed]
Sapbamrer, R.; Hongsibsong, S. Organophosphorus pesticide residues in vegetables from farms, markets,
and a supermarket around Kwan Phayao Lake of Northern Thailand. Arch. Environ. Contam. Toxicol. 2014
7, 60–67. [CrossRef] [PubMed]
,
6
5.
6.
7.
8.
Dar, M.A.; Kaushik, G.; Villarreal-Chiu, J.F. Pollution status and bioremediation of chlorpyrifos in
environmental matrices by the application of bacterial communities: A review. J. Environ. Manag. 2019, 239,
1
24–136. [CrossRef]
Xiong, J.F.; Guan, Z.; Zhou, G.; Tang, X.; Lv, Y.; Wang, H. Determination of chlorpyrifos in environmental
water samples by dispersive liquid-liquid microextraction with solidification of a floating organic drop
followed by gas chromatography with flame photometry detection. Anal. Methods 2012, 4, 3246–3250.
Yin, P.; Guo, G.Y.; Dai, J.X.; Zhu, L.; Gao, G.W.; Chen, H.P.; Liu, X. Determination of chlorpyrifos and its main
metabolite in tea by ultra-high-performance liquid chromatography-tandem mass spectrometry. Chin. J.
Anal. Chem. 2019, 47, 306–314.
Varela-Martinez, D.A.; Gonzalez-Curbelo, M.A.; Gonzalez-Salamo, J.; Hernandez-Borges, J. Analysis of
multiclass pesticides in dried fruits using QuEChERS-gas chromatography tandem mass spectrometry.
Food Chem. 2019, 297. [CrossRef]

Int. J. Environ. Res. Public Health 2020, 17, 4723
12 of 14
9
1
1
.
Ren, Q.; Xia, T. Application of dispersive liquid-liquid microextraction for the analysis of organophosphorus
pesticides in Hawthorn (Crataegus Pinnatifida Var. Major) juice samples. Acta Chromatogr. 2016, 28, 403–414.
CrossRef]
[
0. Garabrant, D.H.; Aylward, L.L.; Berent, S.; Chen, Q.X.; Timchalk, C.; Burns, C.J.; Hays, S.M.; Albers, J.W.
Cholinesterase inhibition in chlorpyrifos workers: Characterization of biomarkers of exposure and response
in relation to urinary TCPy. J. Expo. Sci. Environ. Epidemiol. 2009, 19, 634–642. [CrossRef]
1. Krenz, J.E.; Hofmann, J.N.; Smith, T.R.; Cunningham, R.N.; Fenske, R.A.; Simpson, C.D.; Keifer, M.
Determinants of butyrylcholinesterase inhibition among agricultural pesticide handlers in Washington state:
An update. Ann. Occup. Hyg. 2015, 59, 25–40. [PubMed]
1
2. Basha, P.M.; Poojary, A. Chlorpyrifos induced region-specific vulnerability in rat CNS and modulation by
age and cold stress: An interactive study. Neurochem. Res. 2011, 36, 241–249. [CrossRef] [PubMed]
3. Proskocil, B.J.; Bruun, D.A.; Thompson, C.M.; Fryer, A.D.; Lein, P.J. Organophosphorus pesticides decrease
1
M2 muscarinic receptor function in guinea pig airway nerves via indirect mechanisms. PLoS ONE 2010
, e10562. [CrossRef] [PubMed]
,
5
14. Ismail, A.A.; Wang, K.; Olson, J.R.; Bonner, M.R.; Hendy, O.; Rasoul, G.A.; Rohlman, D.S. The impact
of repeated organophosphorus pesticide exposure on biomarkers and neurobehavioral outcomes among
adolescent pesticide applicators. J. Toxicol. Environ. Health Part A 2017, 80, 542–555. [CrossRef]
15. Lahouel, A.; Kebieche, M.; Lakroun, Z.; Rouabhi, R.; Fetoui, H.; Chtourou, Y.; Djamila, Z.; Soulimani, R.
Neurobehavioral deficits and brain oxidative stress induced by chronic low dose exposure of persistent
organic pollutants mixture in adult female rat. Environ. Sci. Pollut. Res. 2016, 23, 19030–19040. [CrossRef]
16. Fiedler, N.; Rohitrattana, J.; Siriwong, W.; Suttiwan, P.; Strickland, P.O.; Ryan, P.B.; Rohlman, D.S.; Panuwet, P.;
Barr, D.B.; Robson, M.G. Neurobehavioral effects of exposure to organophosphates and pyrethroid pesticides
among Thai children. Neurotoxicology 2015, 48, 90–99. [CrossRef]
1
1
1
2
7. Ismail, A.A.; Bodner, T.E.; Rohlman, D.S. Neurobehavioral performance among agricultural workers and
pesticide applicators: A meta-analytic study. Occup. Environ. Med. 2012, 69, 457–464. [CrossRef]
8. Rohlman, D.S.; Lasarev, M.; Anger, W.K.; Scherer, J.; Stupfel, J.; McCauley, L. Neurobehavioral performance
of adult and adolescent agricultural workers. Neurotoxicology 2007, 28, 374–380. [CrossRef]
9. Cassereau, J.; Ferre, M.; Chevrollier, A.; Codron, P.; Verny, C.; Homedan, C.; Lenaers, G.; Procaccio, V.;
May-Panloup, P.; Reynier, P. Neurotoxicity of insecticides. Curr. Med. Chem. 2017, 24, 2988–3001. [CrossRef]
0. Ramin, M.; Omidi, F.; Khadem, M.; Shahtaheri, S.J. Combination of dispersive solid-phase extraction with
dispersive liquid-liquid microextraction followed by high-performance liquid chromatography for trace
determination of chlorpyrifos in urine samples. J. Environ. Anal. Chem. 2019. [CrossRef]
2
1. Song, S.M.; Huang, H.L.; Chen, Z.J.; Wei, J.; Deng, C.; Tan, H.H.; Li, X.S. Representative commodity for six
leafy vegetables based on the determination of six pesticide residues by gas chromatography. Acta Chromatogr.
2
019, 31, 49–56. [CrossRef]
2
2. Yang, Y.H.; Kong, W.J.; Zhao, L.H.; Xiao, Q.; Liu, H.M.; Zhao, X.S.; Yang, M.H. A multiresidue method for
simultaneous determination of 44 organophosphorous pesticides in pogostemon cablin and related products
using modified QuEchERS sample preparation procedure and Gc-Fpd. J. Chromatogr. B 2015, 974, 118–125.
[CrossRef]
2
3. Zhang, H.T.; West, D.; Shi, H.L.; Ma, Y.F.; Adams, C.; Eichholz, T. Simultaneous determination of
selected trace contaminants in drinking water using solid-phase extraction-high performance liquid
chromatography-tandem mass spectrometry. Water Air Soil Pollut. 2019, 230. [CrossRef]
2
4. Garlito, B.; Ibanez, M.; Portoles, T.; Serrano, R.; Amlund, H.; Lundebye, A.K.; Sanden, M.; Berntssen, M.H.G.;
Hernandez, F. Lc-Ms/Ms method for the determination of organophosphorus pesticides and their metabolites
in salmon and zebrafish fed with plant-based feed ingredients. Anal. Bioanal. Chem. 2019, 411, 7281–7291.
[
CrossRef] [PubMed]
5. Mondal, R.; Mukherjee, A.; Biswas, S.; Kole, R.K. Gc-Ms/Ms determination and ecological risk assessment of
pesticides in aquatic system: A case study in Hooghly River Basin in West Bengal, India. Chemosphere 2018
06, 217–230. [CrossRef] [PubMed]
2
2
,
2
6. Liu, J.L.; Zabetakis, D.; Acevedo-Velez, G.; Goldman, E.R.; Anderson, G.P. Comparison of an antibody and its
recombinant derivative for the detection of the small molecule explosive 2,4,6-Trinitrotoluene. Anal. Chim. Acta
2013, 759, 100–104. [CrossRef]

Int. J. Environ. Res. Public Health 2020, 17, 4723
13 of 14
2
7. Suzuki, C.; Ueda, H.; Mahoney, W.; Nagamune, T. Open sandwich enzyme-linked immunosorbent assay for
the quantitation of small haptens. Anal. Biochem. 2000, 286, 238–246. [CrossRef]
2
8. Wang, K.; Liu, Z.P.; Ding, G.C.; Li, J.; Vasylieva, N.; Li, Q.X.; Li, D.Y.; Gee, S.J.; Hammock, B.D.; Xu, T.
Development of a one-step immunoassay for triazophos using camel single-domain antibody-alkaline
phosphatase fusion protein. Anal. Bioanal. Chem. 2019, 411, 1287–1295. [CrossRef]
2
3
3
3
9. Wang, C.M.; Li, X.B.; Liu, Y.H.; Guo, Y.R.; Xie, R.; Gui, W.J.; Zhu, G.N. Development of a mab-based
heterologous immunoassay for the broad-selective determination of organophosphorus pesticides. J. Agric.
Food Chem. 2010, 58, 5658–5663. [CrossRef]
0. Luo, L.; Song, Y.; Zhu, C.Z.; Fu, S.F.; Shi, Q.R.; Sun, Y.M.; Jia, B.Z.; Du, D.; Xu, Z.L.; Lin, Y.H. Fluorescent
silicon nanoparticles-based ratiometric fluorescence immunoassay for sensitive detection of ethyl carbamate
in red wine. Sens. Actuators B Chem. 2018, 255, 2742–2749. [CrossRef]
1. Hua, X.D.; Qian, G.L.; Yang, J.F.; Hu, B.S.; Fan, J.Q.; Qin, N.; Li, G.; Wang, Y.Y.; Liu, F.Q. Development
of an immunochromatographic assay for the rapid detection of chlorpyrifos-methyl in water samples.
Biosens. Bioelectron. 2010, 26, 189–194. [CrossRef] [PubMed]
2. Zhao, F.C.; Hu, C.Y.; Wang, H.M.; Zhao, L.Y.; Yang, Z.Y. Development of a mab-based immunoassay for the
simultaneous determination of O, O-diethyl and O, O-dimethyl organophosphorus pesticides in vegetable
and fruit samples pretreated with QuEChERS. Anal. Bioanal. Chem. 2015, 407, 8959–8970. [CrossRef]
[PubMed]
3
3
3
3
3
3. Hua, X.D.; Eremin, S.A.; Liu, F.Q.; Wang, M.H. Antibody developments and immunoassays for
organophosphorus chemicals: A review. Curr. Org. Chem. 2017, 21, 2640–2652. [CrossRef]
4. Liu, Y.H.; Zhong, D.L.; Shen, D.Y.; Mo, R.H.; Tang, F.B. Determination of four insecticides in bamboo shoot
by Quechers-Mspd combined with Lc-Ms/Ms. J. Food Sci. Technol. Res. 2014, 20, 563–569. [CrossRef]
5. Manclus, J.J.; Montoya, A. Developmentofanenzyme-linkedimmunosorbentassayfor3,5,6-Trichloro-2-Pyridinol.1.
Production and characterization of monoclonal antibodies. J. Agric. Food Chem. 1996, 44, 3703–3709. [CrossRef]
6. Manclus, J.J.; Primo, J.; Montoya, A. Development of a chlorpyrifos immunoassay using antibodies obtained
from a simple hapten design. J. Agric. Food Chem. 1994, 42, 1257–1260. [CrossRef]
7. Peeters, J.M.; Hazendonk, T.G.; Beuvery, E.C.; Tesser, G.I. Comparison of four bifunctional reagents for
coupling peptides to proteins and the effect of the three moieties on the immunogenicity of the conjugates. J.
Immunol. Methods 1989, 120, 133–143. [CrossRef]
3
8. Hongsibsong, S.; Prapamontol, T.; Suphavilai, C.; Wipasa, J.; Pattarawarapan, M.; Kasinrerk, W. Production
of monoclonal antibody to acaricide dicofol and its derivatives. Hybridoma 2010, 29, 495–500. [CrossRef]
9. Hongsibsong, S.; Wipasa, J.; Pattarawarapan, M.; Chantara, S.; Stuetz, W.; Nosten, F.; Prapamontol„ T.
Development and application of an indirect competitive enzyme-linked immunosorbent assay for the
3
0
detection of p,p -DDE in human milk and comparison of the results against GC-ECD. J. Agric. Food Chem.
012, 60, 16–22. [CrossRef]
2
4
4
0. Vasylieva, N.; Ahn, K.C.; Barnych, B.; Gee, S.J.; Hammock, B.D. Development of an immunoassay for the
detection of the phenylpyrazole insecticide fipronil. Environ. Sci. Technol. 2015, 49, 10038–10047. [CrossRef]
1. Piao, Y.Z.; Kim, Y.J.; Kim, Y.A.; Lee, H.S.; Hammock, B.D.; Lee, Y.T. Development of ELISAS for the
class-specific determination of organophosphorus pesticides. J. Agric. Food Chem. 2009, 57, 10004–10013.
[CrossRef] [PubMed]
4
2. Zhang, Q.; Sun, Q.; Hu, B.S.; Shen, Q.; Yang, G.; Liang, X.; Sun, X.; Liu, F.Q. Development of a sensitive
ELISA for the analysis of the organophosphorous insecticide fenthion in fruit samples. Food Chem. 2008, 106,
1
278–1284. [CrossRef]
4
4
4
3. Hongsibsong, S.; Sapbamrer, R. Removal of organophosphorus pesticide residues in leaf and non-leaf
vegetables by using ozone water. Chiang Mai J. Sci. 2018, 45, 1759–1769.
4. Gabaldon, J.A.; Maquieira, A.; Puchades, R. Development of a simple extraction procedure for chlorpyrifos
determination in food samples by immunoassay. Talanta 2007, 71, 1001–1010. [CrossRef]
5. Kim, Y.A.; Lee, E.H.; Kim, K.O.; Lee, Y.T.; Hammock, B.D.; Lee, H.S. Competitive immunochromatographic
assay for the detection of the organophosphorus pesticide chlorpyrifos. Anal. Chim. Acta 2011, 693, 106–113.
[CrossRef]
4
6. Brun, E.M.; Garces-Garcia, M.; Banuls, M.J.; Gabaldon, J.A.; Puchades, R.; Maquieira, A. Evaluation of a
novel malathion immunoassay for groundwater and surface water analysis. Environ. Sci. Technol. 2005, 39,
2
786–2794. [CrossRef]

Int. J. Environ. Res. Public Health 2020, 17, 4723
14 of 14
4
7. Xu, Z.L.; Deng, H.; Deng, X.F.; Yang, J.Y.; Jiang, Y.M.; Zeng, D.P.; Huang, F.; Shen, Y.D.; Lei, H.T.; Wang, H.;
et al. Monitoring of organophosphorus pesticides in vegetables using monoclonal antibody-based direct
competitive ELISA followed by Hplc-Ms/Ms. Food Chem. 2012, 131, 1569–1576. [CrossRef]
8. Zhao, F.C.; Wang, H.M.; Han, X.; Yang, Z.Y. Development and comparative study of chemosynthesized
antigen and mimotope-based immunoassays for class-specific analysis of O,O-dimethyl organophosphorus
pesticides. Sci. Rep. 2016, 6, 37640. [CrossRef]
4
4
5
5
9. Li, M.; Sheng, E.; Cong, L.; Wang, M. Development of immunoassays for detecting clothianidin residue in
agricultural products. J. Agric. Food Chem. 2013, 61, 3619–3623. [CrossRef]
0. Mitake, S. Development of immunoassay test kit “Smartassay Series” for pesticide analysis. In Horiba
Technical Reports; Horiba: Fukuoka, Japan, 2012; Volume 16.
1. Vasylieva, N.; Barnych, B.; Rand, A.; Inceoglu, B.; Gee, S.J.; Hammock, B.D. Sensitive immunoassay for
detection and quantification of the neurotoxin, tetramethylenedisulfotetramine. Anal. Chem. 2017, 89,
5
612–5619. [CrossRef]
5
5
2. Prasopsuk, J.; Iwai, C.B. Risk assessment of pesticide residues in organic waste in Northeast Thailand. Int. J.
Environ. Rural Dev. 2011, 2, 5.
3. Wanwimolruk, S.; Phopin, K.; Boonpangrak, S.; Prachayasittikul, V. Food safety in Thailand 4: Comparison
of pesticide residues found in three commonly consumed vegetables purchased from local markets and
supermarkets in Thailand. PeerJ 2016, 4, e2432. [CrossRef] [PubMed]
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(