Development of an enzyme-linked immunosorbent assay for the pyrethroid insecticide cyhalothrin

Article abstract of DOI:10.1021/jf0607009

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for detection of the pyrethroid insecticide cyhalothrin. Three haptens with an amine or propanoic acid terminus were synthesized and then conjugated with bovine serum albumin to give immunogens. Eight polyclonal antisera produced by rabbits were screened for titers and affinity using three different coating antigens. The antiserum CWB-C had the highest affinity with cyhalothrin and a low affinity with fenvalerate, fenpropathrin, deltamethrin, and fluvalinate. The half-maximum inhibition concentration for cyhalothrin was 37.2 μg/L, and the limit of detection was 4.7 μg/L. The recoveries of different concentrations of cyhalothrin (0.1-2500 μg/L) from fortified tap water, well water, and wastewater samples as determined with the ELISA were 81-114%.

Full text of DOI:10.1021/jf0607009

5284 J. Agric. Food Chem. 2006, 54, 5284−5291
Development of an Enzyme-Linked Immunosorbent Assay for
the Pyrethroid Insecticide Cyhalothrin
†
‡
†
‡
†
HONGBIN GAO, YUN LING, TING XU, WEIWEN ZHU, HONGYU JING,
†
§
,†
WEI SHENG, QING X. LI, AND JI LI*
Department of Ecology and Environmental Sciences, College of Resource and
Environment and College of Natural Sciences, China Agricultural University,
Beijing, 100094, People’s Republic of China, and Department of Molecular Biosciences and
Bioengineering, University of Hawaii, Hawaii 96822
A competitive enzyme-linked immunosorbent assay (ELISA) was developed for detection of the
pyrethroid insecticide cyhalothrin. Three haptens with an amine or propanoic acid terminus were
synthesized and then conjugated with bovine serum albumin to give immunogens. Eight polyclonal
antisera produced by rabbits were screened for titers and affinity using three different coating antigens.
The antiserum CWB-C had the highest affinity with cyhalothrin and a low affinity with fenvalerate,
fenpropathrin, deltamethrin, and fluvalinate. The half-maximum inhibition concentration for cyhalothrin
was 37.2 µg/L, and the limit of detection was 4.7 µg/L. The recoveries of different concentrations of
cyhalothrin (0.1-2500 µg/L) from fortified tap water, well water, and wastewater samples as determined
with the ELISA were 81-114%.
KEYWORDS: ELISA; pyrethroid; cyhalothrin
INTRODUCTION
flucythrinate (19), permethrin (20, 21), and phenothrin (22). An
immunoassay was developed for cyhalothrin by Lee et al. (16).
Because Lee et al. intended to develop a class-specific assay
for type II pyrethroids, the cross-reaction of the deltamethrin
immunoassay was 40-700% for cyhalothrin detection, and the
assay was not applied for the analysis of environmental samples
Cyhalothrin, registered by the U.S. Environmental Protection
Agency in 1988 (1), is a synthetic pyrethroid and is widely used
for the control of many common pests on crops and domestic
animals (2, 3). In China, cyhalothrin is widely used on cotton,
teas, fruit trees, and vegetables. However, cyhalothrin is highly
toxic when it is ingested. The acute oral LD50 values in rats
and mice are 79 and 56 mg/kg, respectively (4). Cyhalothrin is
especially highly toxic to fish (LC50, 0.078-2.3 µg/L) and
aquatic invertebrates (LC50, 0.0023-3.3 µg/L). The half-life of
cyhalothrin is 30, 40, and 23 days in soil, plants, and fat tissues,
respectively (4-6). Laboratory studies indicated that cyhalothrin
could be bioaccumulated in fish and that it had an adverse
impact on human health (4, 7). Thus, a sensitive, selective, and
rapid method for monitoring residue levels of cyhalthrin in
aquatic ecosystem is desirable.
The reported methods for detection of cyhalothrin include
high-performance liquid chromatography (HPLC) (8), gas chro-
matography with electron capture detection (GC-ECD) (9), and
GC-mass spectrometry (GC-MS) (10). These methods are ex-
pensive and labor intensive and require extensive analytical
skills. Immunoassays have been proven to be a fast, sensitive,
and selective method for detection of pesticides at a trace level
(16). A cyhalothrin-specific antibody will advance the study of
cyhalothrin environmental fate and can be used in antibody mi-
croarray or immunoaffinity chromatography. The objective of
this work was to develop a sensitive and selective immunoassay
for the detection of cyhalothrin in tap water, well water, and
wastewater.
MATERIALS AND METHODS
Reagents. Cyhalothrin, fenvalerate, fenpropathrin, deltamethrin,
fluvalinate, and cypermethrin were obtained from China National
Research Center for Certified Reference Materials (Beijing, China).
All organic starting materials for hapten synthesis were purchased form
Aldrich Chemical Co. (Milwaukee, WI). The coupling reagents goat-
anti-rabbit (GAR) immunoglobin conjugated to horseradish peroxidase
(HRP), bovine serum albumin (BSA), ovalbumin (OVA), and tetra-
methylbenzidine (TMB) were purchased from Sigma Chemical Co. (St.
Louis, MO).
Apparatus. GC analysis was performed on a GC-9900 equipped
with a flame ionization detector (Beijing Jiafen Analitical Instrument
Factory, China). Separations were accomplished on a homemade
capillary GC SE-54 column (40 m × 0.5 mm, 0.25 µm). The
temperature of the injector port and the detector was 250 °C. The oven
temperature was 230 °C. The carrier was nitrogen at a flow rate of 20
mL/min. Nuclear magnetic resonance (NMR) spectra was obtained with
a General Electic QE-300 spectrometer. Chemical shift values were
given in parts per million downfield from internal tetramethylsilane.
Enzyme-linked immunosorbent assays (ELISAs) were carried out in
(
11). Several immunoassays have been well-developed for the
detection of synthetic pyrethoids such as allethrin (12), (s)-
bioallethrin (13, 14), bioresmethrin (15), cypermethrin (16),
deltamethrin (16, 17), esfenvalerate (11), fenpropathrin (18),
*
To whom correspondence should be addressed. Tel: +86-10-62819331.
E-mail: liji@cau.edu.cn.
†
College of Resource and Environment, China Agricultural University.
‡
College of Natural Sciences, China Agricultural University.
§
University of Hawaii.
1
0.1021/jf0607009 CCC: $33.50
© 2006 American Chemical Society
Published on Web 06/23/2006

ELISA for the Pyrethroid Insecticide Cyhalothrin
J. Agric. Food Chem., Vol. 54, No. 15, 2006 5285
Scheme 3. N-1, N-2, N-3, and N-4
Scheme 1. Synthesis of Cwb-1, Cwb-2, Cwb-3, Cwb-4, Cwb-5, and
Cwb-6
ambient temperature, the mixture was diluted with ether, washed with
a sodium carbonate solution followed by a water wash, and dried with
anhydrous MgSO
4
. Solvents were evaporated, and the product was
distilled under vacuum to yield 13.5 g, 57.9%, of Cwb-2 as a colorless
1
oil. H NMR (CDCl
.20-7.63 (4H, m, ArH).
Step C. Benzyl 4-(3-formylphenoxy)benzenepropanoate (Cwb-3) was
formed by following the procedures of Wengatz et al. (18). Potassium
tertbutoxide (65 mL, 1 M in tertbutyl alcohol) was added under N to
3 3 2
): 3.31, [6H, s, (CH O) ], 5.36, (1H, s, phCH),
7
2
a stirred solution of Cwb-1 (16.6 g, 64.8 mmol) in 180 mL of xylene.
The mixture was heated to remove 100 mL of distillate. An additional
Scheme 2. Synthesis of Dwb-1, Dwb-2, Dwb-3, Dwb-4, and Dwb-5
4
1
0 mL of xylene was added and distilled to a kettle temperature of
36 °C. The resulting salt suspension was cooled and treated with
pyridine (16 mL), cuprous chloride (0.6 g), copper powder (0.3 g),
8-crown-6 (100 mg), and 15.0 g (65 mmol) of Cwb-2. This mixture
was refluxed under N for 17 h, cooled, washed with water, and filtered
through silica gel (25 g). The pad of silica gel was washed with CH
Cl (40 mL). The combined filtrate and washing were evaporated to a
volume of 30 mL, diluted with wet CH Cl (40 mL), and treated with
acidified silica gel (30 g), previously treated in ether with 9 drops of
8% H SO and stripped to a dry powder on a rotary evaporator. After
1
2
2
-
2
2
2
9
2
4
water (1 mL) was added, this mixture was stirred for 2 h. After filtration
and evaporation, the mixture was flash-chromatographed on a silica
2 2
gel column (200 g), eluting with hexane/CH Cl (80:20 f 0:100).
Stripping at 30 °C and 0.1 Torr yielded 13.1 g (56%) of Cwb-3 as a
1
pale yellow oil. H NMR (CDCl
.99 (2H, t, phCH CH ), 5.12 (3H, s, phCH
ArH), 9.93 (1H, s, CHO).
Step D. Cwb-3 (1.3 g, 3.4 mmol) in 16 mL of tetrahydrofuran (THF)
was added to a stirred solution of 1.5 g of KCN in 4 mL of H
precooled with ice followed by slow addition of 4 mL of H SO (40%).
After 30 min and on ice, the mixture was extracted with ether three
times and dried with anhydrous MgSO . After the solvent was removed,
3
): δ 2.66-2.71 (2H, t, COCH
2
), 2.94-
2
2
2
2
O), 6.92-7.59 (8H, m,
9
6 well microtiter plates, and the absorbance was read with a Vmax
microplate reader (Molecular Devices, Menlo Park, CA).
Hapten Synthesis and Verification. Haptens were synthesized as
outlined in Schemes 1-3.
2
O
2
4
Step A. Benzyl 4-hydroxybenzenepropanoate (Cwb-1) was synthe-
sized according to the procedures of Wengatz et al. (18). A mixture of
4
1.1 g of benzyl 3-[3-(cyano-hydroxy-methyl)phenoxy]benzenepro-
4
-hydroxybenzenepropanoic acid (14.4 g, 86.6 mmol), benzyl alcohol
panoate (Cwb-4) was obtained in 77% yield, which was a pale solid.
1
(27 mL), toluene (15 mL), and 4 drops of 85% phosphoric acid was
H NMR (CDCl
3
): δ 2.63-2.68 (2H, t, COCH
2
), 2.88-2.94 (2H, t,
O), 5.40
heated to reflux under a dean-stark trap. Toluene was removed up to a
kettle temperature of 145-150 °C. Heating was continued for 10 h to
collect 1.50 mL of water. Excess benzyl alcohol was removed under
vacuum to a kettle temperature of 150 °C. The residue was dissolved
phCH
2
CH ), 4.26 (1H, s, b, CNCHOH), 5.07 (2H, s, phCH
2
2
(1H, s, CNCH), 6.87-7.34 (13H, m, ArH). Following the above
procedure for Cwb-4, the reaction of Dwb-2 (4.9 g, 18.2 mmol) instead
of Cwb-3 gave 5.2 g of [3-(cyano-hydroxy-methyl)phenoxy]acetic acid
1
in ether, washed, and dried with anhydrous MgSO
was distilled through a short path heated from an oil bath at 230-240
C to collect 16.1 g (73%) of product at a heat temperature of 185-
4
. The stripped residue
benzyl ester (Dwb-3) as a white solid; yield, 96%. H NMR (CDCl
3
):
),
2 2
δ 2.90 (1H, s, b, CNCHOH), 4.69 (2H, s, CH CO), 5.24 (2H, s, phCH
°
1
5.46 (1H, s, CNCH), 6.92-7.37 (9H, m, ArH). The reaction of N-1
1
94 °C. H NMR (CDCl
2H, t, phCH CH ), 5.09 (2H, s, phCH
.35 (9H, m, ArH).
Step B. Bromo-3-(dimethoxymethyl)benzene (Cwb-2) was obtained
3
): δ 2.60-2.67 (2H, t, COCH
2
), 2.85-2.89
(1.68 g, 6.9 mmol) gave 2.34 g of 2-hyroxy-2-[3-(4-nitrophenoxy)-
1
(
7
2
2
2
O), 6.23 (1HS, b, phOH), 6.69-
phenyl]acetonitrile (N-2) as a brown sticky liquid; yield, 89%. H NMR
(CDCl
Step E. Dicyclohexylcarbodiimide (DCC) (1.3 g, 6.1 mmol) in 15
mL of CH Cl was dropped into a stirred solution of Cwb-4 (2.4 g, 6.4
mmol) and (()-(cis)-3-[(Z)-3-chloro-4,4,4-trifluorobut-1-enyl-2,2-dim-
ethyl]cyclopropane carboxylic acid (1.3 g, 5.5 mmol) in 15 mL of CH
Cl with ice cooling. After DCC in 15 mL of CH Cl was added for
3
): δ 5.83-5.85 (1H, d, CNCH), 7.15-8.31 (9H, ArH, CHOH).
according to the procedure of Wengatz et al. (18). Two drops of
concentrate sulfuric acid were added to a solution of 3-bromobenzal-
dehyed (18.5 g, 0.1 mol) and trimethylorthoformate (12.2 g, 0.1 mol)
in 15 mL of methanol. After an immediate mild exotherm and 3 h at
2
2
2
-
2
2
2

5286 J. Agric. Food Chem., Vol. 54, No. 15, 2006
Gao et al.
about 2 h, the mixture was concentrated. Chromatographic purification
on silica (R ) 0.5, ethyl acetate:petroleum ether, 1:5) gave 2.5 g of
benzyl-3-[(()cyano[(()-(cis)-3-[(Z)-3-chloro-4,4,4-trifluorobut-1-enyl-
,2-dimethyl]cyclopropanecarbonyloxy]phenoxy]benzenepropanoate
Cwb-5) as a straw yellow sticky liquid; yield, 75%. 1H NMR
CDCl ): δ 1.28-1.33 [6H, t, (CH C], 2.01-2.05 (1H, d, CHCO),
.49-2.31 (1H, t, dCHCH), 2.67-2.72 (2H, t, COCH ), 2.95-2.99
2H, t, phCH CH ), 5.12 (2H, s, phCH O), 6.31, 6.37 (1H, s, CNCH),
.80-6.81 (1H, d, dCH), 6.92-7.39 (13H, m, ArH). Following the
above procedure for Cwb-5, the reaction of DCC (1.7 g, 8.2 mmol),
Dwb-3 (2.2 g, 7.4 mmol), and (()-(cis)-3-[(Z)-3-chloro-4,4,4-trifluo-
robut-1-enyl-2,2-dimethyl]cyclopropane carboxylic acid (2.41 g, 9.9
mmol) gave 2.57 g of 3-[(()cyano[(()-(cis)-3-[(Z)-3-chloro-4,4,4-
trifluorobut-1-enyl-2,2-dimethyl]cyclopropanecarbonyloxy]phenoxy]-
K
2
CO
3
in 11.5 mL of DMF. The mixture was heated to reflux at 150
f
°C for 10 h and then poured into 200 mL of ice water. A brown solid
was formed. The brown solid was dissolved completely in the solution
(ethyl acetate:petroleum ether ) 1:1) when heated. The solution was
kept in 4 °C overnight. After filtration, 2.1 g of 3-(4-nitrophenoxy)-
benzaldehyde (N-1) as a yellow solid was obtained; yield, 87%. H
3
NMR (CDCl ): δ 7.05-8.26 (8H, m ArH), 10.02 (1H, s, CHO).
Step J. (()Cyano 3-(4-aminophenoxy)phenyl-(()-cis-3-(Z-2-chloro-
3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcycloproanecarboxylate (N-4)
was obtained according to the procedures of Shan et al. (11). Stannous
chloride hydrate (1.4 g, 6.2 mmol) was added to a stirred solution of
N-3 (0.6 g, 1.2 mmol) in 0.75 mL of ethanol (EtOH), and the mixture
2
(
(
2
(
1
3
3
)
2
2
2
2
2
6
was heated under N at 70-75 °C for 30 min. The mixture was cooled
and poured into a slurry of water, ether, and Celiet (0.4 g). NaHCO3
2
acetic acid benzyl ester (Dwb-4), yield 62%, which was a pea green
(1.4 g, 1.2 mmol) was added in portions and stirred until CO evolution
2
1
sticky liquid. H NMR (CDCl
3
): δ 1.23-1.34 [6H, t, (CH
3
)
2
C], 2.00-
ceased. The mixture was filtered, and solids were extracted with ether.
Combined ether extracts were stripped, and the residue was chromato-
graphed on a 5 g silica gel column (50% CH Cl /hexane f CH Cl )
2
2
.04 (1H, dd, CHCO), 2.21-2.28 (1H, q, dCHCH), 4.69 (2H, s, CH -
CO), 5.25 (2H, s, phCH ), 6.30-6.36 (1H, d, CNCH), 6.81-6.85 (1H,
2
2
2
2
2
d, dCH), 6.94-7.38 (9H, m, ArH). The reaction of N-2 (1.6 g, 5.9
mmol), (()-(cis)-3-[(Z)-3-chloro-4,4,4-trifluorobut-1-enyl-2,2-dimethyl]-
cyclopropane carboxylic acid (1.41 g, 5.3 mmol) and DCC (1.22 g,
to give 0.4 g of N-4, yield 70%, as a colorless viscous gum. TLC Rf
0.3 (CH Cl ). The TLC spot rapidly darkened on exposure to sunlight.
2
2
1
H NMR (CDCl ): δ 1.22-1.39 [6H, t, (CH ) C], 2.01-2.05 (1H, d,
3
3 2
5
(
.9 mmol) gave 0.61 g of (()cyano[3-(4-nitrophenoxy)phenyl]methyl-
()-cis-3-(Z-2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopro-
pane carboxylate (N-3), yield 21%, which was a brown sticky liquid.
COCH), 2.21-2.27 (1H, t, dCHCH), 3.60 (2H, s, b, NH ), 6.28-6.35
(1H, d, CNCH), 6.68-6.70 (1H, d, dCH), 6.70-7.37 (8H, m, ArH).
2
Hapten Conjugation. Conjugates were synthesized according to
water-soluble carbodiimide and diazotization methods (11). Immuno-
gens were conjugates of haptens N-4, Cwb-6, or Dwb-5 with BSA.
Coating antigens were conjugates of haptens Cwb-6, N-4, or Dwb-5
with OVA.
Polyclonal Antibody Production. Female New Zealand white
rabbits were injected subcutaneously five times with 1 mg of BSA-
hapten conjugate at 2 week intervals. The first injection consisted of 1
mL of the conjugate mixed with 1 mL of complete Freund’s adjuvant.
Incomplete adjuvant was used instead of complete in the subsequent
boost injections. The rabbits were bled from the ear vein 10 days after
each injection except for the initial injection.
1
H NMR (CDCl
3
3 2
): δ 1.24-1.35 [6H, m, (CH ) C], 2.04-2.07 (1H, d,
CHCO), 2.24-2.31 (1H, q, dCHCH), 6.38-6.43 (1H, d, CNCH),
6
.79-6.83, (1H, d, dCH), 7.03-8.26 (8H, m, ArH).
Step F. 3-[(()Cyano[(()-(cis)-3-[(Z)-3-chloro-4,4,4-trifluorobut-1-
enyl-2,2-dimethyl]cyclopropane carbonyloxy]phenoxy]propanoic acid
(
Cwb-6) and 3-[(()cyano[(()-(cis)-3-[(Z)-3-chloro-4,4,4-trifluorobut-
-enyl-2,2-dimethyl]cyclopropane carbonyloxy]phenoxy]acetic acid
Dwb-5) were synthesized according to the procedure of Wengatz et
al. (18). The ester Cwb-5 (1.4 g, 2.3 mmol) in 1.5 mL of dry CH Cl
1
(
2
2
was treated with 20 µL of bis(trimethylsilyl)trifluoroacetamide (BSTFA)
followed by iodotrimethylsilane (TMSI, 0.18 mL). After 1.5 h, 5 drops
of water and 1 mL of CH
for 5 min. The organic phase was immediately flash-chromatographed
on 10 g of silica gel G450 (CH Cl f ethyl acetate). The product was
2
Cl
2
were added and the mixture was stirred
Enzyme Immunoassay. The method was identical to that reported
by Shan et al. (11) with the following modifications. The blocking
solution was 0.5% OVA in phosphate-buffered saline (PBS). The
incubation step with goat anti-rabbit-IgG-HRP conjugate or analytes
was 30 min at 37 °C. The reaction of color was at 37 °C for 5-10
min. Standard curves were obtained by plotting absorbance against the
logarithm of analyte concentration. The curves were fitted to a four-
2
2
stripped from a thin-layer chromatography (TLC) plate (<1 mm) to
1
yield 0.9 g of Cwb-6, yield 76%, which was a pale yellow gum. H
NMR (CDCl
3
): δ 1.31 [6H, s, (CH
3
)
2
C], 2.01-2.06 (1H, d, CHCO),
CO), 2.93-2.98 (2H,
), 6.31 (1H, s, CNCH), 6.82-6.85 (1H, d, dCH), 6.93-7.40
2
.22-2.28 (1H, t, dCH), 2.66-2.71 (2H, t, CH
2
t, phCH
2
parameter logistic equation: y ) {(A - D)/ [1 + (x/C) ]} + D. A is
B
(8H, m, ArH), 10.50 (1H, S, b, COOH). Following the above procedure
the maximum absorbance at no analyte present. B is the curve slop at
the inflection point. C is the concentration of analyte giving 50%
inhibition. D is the minimum absorbance at infinite concentration of
for Cwb-6, the reaction of Dwb-4 (2.6 g, 4.8 mmol) gave 0.8 g of
1
Dwb-5, yield 35%, which was a yellow liquid. H NMR (CDCl
.19-1.29 [6H, t, (CH C], 1.97-2.01 (1H, d, CHCO), 2.18-2.24
1H, t, dCHCH), 4.38 (2H, s, CH ), 6.19-6.24 (1H, d, CNCH), 6.79-
.82 (1H, d, dCH), 6.82-7.19 (4H, m, ArH).
Step G. Bromoacetic acid (1.1 g, 7.8 mmol) was added to a stirring
solution of 0.6 g of NaOH in 4.8 mL of H O in a 50 mL vessel cooled
3
): δ
1
(
6
3
)
2
the analyte. The I50 value and the limit of detection (LOD) (I ) were
15
2
found through the logistic equation.
Antiserum Characterization and Assay Optimization. After an
immune procedure, eight antisera were procured. However, the anti-
serum N-2 was hemolytic and not good to used in the immunoassay.
Titers of other seven antisera were tested against three different coating
antigens according to the procedure of Shan et al. (11). All seven
antisera showed higher titers in a homologous system than those in
the heterologous system, which was consistent with the findings of
Shan et al. (11) and Wengatz et al. (18). The titers of different antisera
varied with immnogens and coating antigens. The titer of CWB-C/
Cwb-OVA (antiserum/coating antigen) was the highest titer among all
of the tested combinations of the seven antisera with different coating
antigens. The homologous systems, whose absorbance was higher than
0.75, were tested for cyhalothrin inhibition (Table 1). The I50 values
ranged from 56 to 107 µg/L (Table 2), and the CWB-C/Cwb-OVA
was the lowest I50, which was chosen to be further optimized. Antisera
dilution and coating antigens concentrations were further optimized in
a two-dimensional titration (Table 3). The combination of antiserum
dilution/coating antigen dilution (4000/1000) giving the highest value
was selected for further assay development.
2
with an ice bath. After 3-hydroxybenzaldehyde (0.9 g, 7.4 mmol) was
added, the solution was refluxed for 2 h. The mixture was cooled with
an ice bath, and the pH was adjusted with HCl to pH 1. Filtration under
vacuum and then recrystallization in water gave 0.9 g of (3-formyl-
1
phenoxy)acetic acid (Dwb-1); yield, 68%. H NMR (CDCl
3
): δ 4.79
(
2H, s, CH
2
), 7.25-7.56 (4H, m, ArH), 9.98 (1H, s, CHO), 13.10 (1H,
s, b, COOH).
Step H. 1-(Chloromethyl)benzene (3.8 g, 30 mmol) and triethylamine
(1.3 g, 12.5 mmol) were added to a stirred solution of Dwb-1 (4.5 g,
2
5 mmol) and NaH (1.2 g, 50%) in 30 mL of dried N,N-dimethylfor-
mamide (DMF). After the solution refluxed at 150 °C for 5 h, the
solution was cooled, diluted with 100 mL of ethyl acetate, washed with
water five times, and then dried by adding anhydrous MgSO into the
4
collected organic phase overnight. Chromatographic purification on
silica (G450, ethyl acetate:petroleum ether ) 1:6) and solvent removal
gave 3.4 g of 3-formyl-phenoxy acetic acid benzyl ester (Dwb-2); yield,
1
5
1%. H NMR (CDCl
3
): δ 4.74 (2H, s, CH
2
CO), 5.25 (2H, s, phCH
2
O),
After the optimizing procedure, the conditions of the ELISA for
cyhalothrin were found, and the inhibition curve was found (Figure
1). The I50 and LOD were 37.2 and 4.7 µg/L, respectively, and the
calibration linear range was 5-500 µg/L.
7
.20-7.53 (9H, m, ArH), 9.94 (1H, s, CHO).
Step I. 3-Hydroxy-benzaldehyde (1.6 g, 14.4 mmol) and 1-choro-
4
-nitrobenzene (1.2 g, 7.7 mmol) were added to a solution of 1.4 g of

ELISA for the Pyrethroid Insecticide Cyhalothrin
J. Agric. Food Chem., Vol. 54, No. 15, 2006 5287
Table 1. Titration Summary for Anti-cyhalothrin Antibodya
coating antigen (1 µg/mL)
antiserum (1:6000)/
immunogen
Cwb-OVA
Dwb-OVA
N-OVA
CWB-A/Cwb-BSA
CWB-B/Cwb-BSA
CWB-C/Cwb-BSA
DWB-A/Dwb-BSA
DWB-B/Dwb-BSA
DWB-C/Dwb-BSA
N-1/N-BSA
+++
+++
++++
++
+
++
++
++
+
++
+++
++
+
+
++++
+++
++++
+++
+++
+
++
a
Absorbance:
+, <0.25; ++, )0.25−0.50; +++, )0.50−0.75; ++++, >0.75.
Goat anti-rabbit IgG-HRP dilution, 1/1000.
Table 2. Selected Competitive ELISA Results for Screening
antiserum
1:6000)
coating antigen
(1 g/mL)
I
50
a
b
R 2
(
µ
A
max
A
min
(
µ
g/L)
CWB-C
DWB-A
N-1
Cwb-OVA
Dwb-OVA
N-OVA
1.21
0.97
1.08
0.15
0.26
0.16
55.6
106.8
87.3
0.99
0.99
1.0
Figure 1. Inhibition curve of the optimized ELISA for cyhalothrin. Coating
antigen (Cwb-OVA) concentration, 1/1000 (0.7
C) dilution, 1/4000; and goat anti-rabbit IgG-HRP dilution, 1/1000. The
values are the means SD of three replicates. The cyhalothrin analyte
µg/L); antiserum (CWB-
a
Maximum absorbance of no analyte control. b Minimum absorbance of 250
mg/L cyhalothrin. Goat anti-rabbit IgG-HRP dilution, 1/1000.
±
standards were prepared in a 50% methanol/PBS solution. The pH value
was 7.0.
Table 3. Optimization of Coating Antigen Concentrations and
Antiserum Dilutions
coating antigen
3
column (6 cm /500 mg, part 10188-1356AB) was obtained from Agilent
0
B −
Ba
1:2000b
(Cwb-OVA) concn
Technologies Co. Ltd. (China). All of the three water samples (tap
water, well water, and wastewater) were spiked with four different
concentrations of cyhalothrin (0, 0.1, 0.5, and 5 µg/L) and then extracted
(µ
g/L)
1:500b
1:1000b
1:4000b
1:8000b
0.43a
0.52
0.61
0.49
3
1
0
0
.5
.4
.7
.35
0.58
0.70
0.85
0.68
0.85
1.12
1.43
1.02
0.91
1.21
1.65
1.17
0.65
0.82
0.92
0.77
2
by SPE. The final elute was evaporated under a gentle N stream, and
then, the residue was reconstituted in 1 mL of methanol and divided
into two aliquots. To one aliquot was added an equal volume of PBS
solution and analyzed by ELISA in a blind fashion; to the other was
added an equal volume of methanol and analyzed by GC in a blind
fashion.
a
B
0
is the maximum absorbance at no analyte present. B is the inhibition
b
absorbance at 250 mg/L cyhalothrin. Antiserum (CWB-C) dilution. Goat anti-rabbit
IgG-HRP dilution, 1/1000.
RESULTS AND DISCUSSION
Effects of solvents, ionic strength, and pH were tested by running
the assay in PBS, PBS at pH 3-9, PBS containing various percents of
methanol (0, 10, 20, 50, 80, and 100%) or acetone (0, 10, 20, and
Hapten Synthesis. There are 16 theoretical cyhalothrin
isomers. Commercial cyhalothrin insecticide products typically
contain four isomers that are (()cyano-3-(4-phenoxyphenyl)-
4
0%), and PBS containing different NaCl concentrations (0.2, 1, 2, 5,
and 8%).
Cross-Reactivity. Cyhalothrin, fenvalerate, fenpropathrin, delta-
(()-cis-3-(Z-2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethyl-
cycloproane carboxylate [Z-(1R,3R)-cis,S; Z-(1S,3S)-cis,R;
Z-(1R,3R)-cis,R; Z-(1S,3S)-cis,S]. Cyhalothrin haptens were
designed to mimic the four cyhalothrin isomers. The (()-(cis)-
methrin, fluvalinate, and cypermethrin were tested for cross-reactivity
by preparing each compound in 50% methanol in PBS and determining
the I50 in the ELISA. Cross-reactivity values were calculated as follows:
3-[(Z)-3-chloro-4,4,4-trifluorobut-1-enyl-2,2-dimethyl]cyclopro-
CR% ) (I50 of cyhalothrin/I50 of tested compound) × 100
panecarboxylic acid moiety was kept as a major part of the
haptens. To keep the (()-(cis)-3-[(Z)-3-chloro-4,4,4-trifluorobut-
Fortification and Recovery of Water Samples. Well water, tap
water, and wastewater were from China Agricultural University and
XiaoQing River. Six pretreatments were designed for each sample. In
pretreatment 1, each water sample was spiked with cyhalothrin at 100
µg/L and then diluted with an equal volume of methanol. The antiserum
solution was 2-fold concentrated so the final concentration of PBS in
the well was 1×. In pretreatment 2, each water sample was spiked
with cyhalothrin at 500 µg/L and then diluted 10 times by PBS with
1-enyl-2,2-dimethyl]cyclopropanecarboxylic acid moiety in hap-
tens from isomerizing during synthesis, a two-step approach
was used. Cwb-3, Dwb-2, and N-1 were converted to Cwb-4,
Dwb-3, and N-2, respectively, followed by conversion to
corresponding Cwb-5, Dwb-4, and N-3 using DCC.
Pyrethroid insecticides differ from their cyclopropane end of
the structures. Therefore, haptens were designed to have a
functional group at the aromatic moiety of the cyhalothrin
molecule for coupling with protein. The cyclopropane end of
the hapten molecules is distal to the conjugate target to elicit
cyhalothrin-specific antibodies. Haptens Cwb-6, Dwb-5, and N-4
were conjugated with BSA to give the immunogens Cwb-BSA,
Dwb-BSA, and N-BSA, respectively, and conjugated with OVA
to give the corresponding coating antigens Cwb-OVA, Dwb-
OVA, and N-OVA.
5
0% methanol. In pretreatment 3, each water sample was spiked with
cyhalothrin at 2500 µg/L and then diluted 50 times by PBS with 50%
methanol. Pretreatments 4-6 were the same as pretreatments 1-3
except that the antiserum PBS solution contained 0.2% bovine blood
fibrinogen.
Solid Phase Extraction (SPE). A C18 SPE method was used for
extraction of cyhalothrin from water samples, which was the same as
the previously reported method for the extraction of esfenvalerate from
water (11). This method did well in the extraction of pyrethroud
insecticide from water, such as fenpropethrin (18), permethrin (20),
deltamethrin (23), and esfenvalerate (11). In this study, the C18 SPE
Optimization. Effects of methanol or acetone on the ELISA
using the antiserum CWB-C and the coating antigen Cwb-OVA

5288 J. Agric. Food Chem., Vol. 54, No. 15, 2006
Gao et al.
Figure 2. Effects of solvents, pH and anionic strength on the ELISA for cyhalothrin. Coating antigen (Cwb-OVA) concentration, 1/1000 (0.7
µg/L);
antiserum (CWB-C) dilution, 1/4000; and goat anti-rabbit IgG-HRP dilution, 1/1000. (a) ELISA competition curves for cyhalothrin in PBS buffer containing
various concentrations of methanol. The concentrations of methanol shown in this figure are the methanol concentrations of the inhibitor solution before
it was added into the well of a 96 well plate. (b) ELISA competition curves for cyhalothrin in PBS buffer containing various concentrations of acetone.
The concentrations of acetone shown in this figure are the acetone concentrations of the inhibitor solution before it was added into the well of a 96 well
plate. (c) ELISA competition curves for cyhalothrin in solutions at various pH values. The average of SD
cyhalothrin in buffers with various ionic strengths. The average of SD 2.7.
) 3.1. (d) ELISA competition curves for
)
are shown in Figure 2a,b, respectively. The results show that
the solvents significantly reduce the ELISA sensitivities.
Methanol or acetone enhanced the binding of antibody-hapten
and kept cyhalothrin soluble in the solution, which was
consistent with the results of fenpropathrin (18). However,
excess high concentrations of solvents would affect the iden-
tification and binding of the antibody-hapten and, furthermore,
denature protein reagents. The I50 of 50% methanol was the
lowest I50, which was two times lower than that of 80%
methanol and 50% acetone.
To estimate potential interferences resulting from pH and salt
concentration variations in environmental water samples, the
effects of pH and ionic strength on the ELISA performance were
evaluated in this study. The pH effects on the ELISA (CWB-
C/Cwb-OVA) are shown in Figure 2c. These results showed
that this ELISA was stable in the range of pH 5.0-8.0 and
higher or lower pH values significantly increased the I50 values,
which was similar to the trends of pH effects on deltamethrin
significantly not only the I50 of cyhalothrin but also the control
absorbance. The optimum range of NaCl concentration was 0.2
f 2.0%.
Cross-Reactivity. The ELISA using the antiserum CWB-C
and the coating antigen Cwb-OVA was selective for cyhalothrin.
Most of the tested compounds showed no significant cross-
reactivity in a range of 0.1-10000 µg/L except fenpropathrin
(CR, <0.05%) and cypermethrin (CR, 2.5%) (Table 4). In this
study, the haptens derived from the aromatic moiety of the
cyhalothrin molecule, which was different from the haptens in
the research of Lee et al. (16), used the hapten deriving from
the cyano of cyhalothrin molecule as the immunogen. Using
the haptens similar to this study, the ELISA selective for
esfevalerate (11), the ELISA selective for deltamethrin (16, 17),
and the ELISA selective for permethrin were found.
Recovery of Water Samples. A rapid and efficient SPE
procedure was used to extract and concentrate cyhalothrin in
water samples for its detection at sub-part per billion levels.
When 200 mL of water sample was extracted with SPE and
analyzed by ELISA and GC, no matrix effect was measured.
All recoveries from the water samples were >80% of the spiked
values (Table 5). Because pyrethroids were hydrophobic
(23) and flucythrinate (19) ELISAs. The assay buffer, pH 7.0,
was stable and most sensitive.
The effects of ionic strength on the ELISA (CWB-C/Cwb-
OVA) are shown in Figure 2d. Increasing salinity decreased

ELISA for the Pyrethroid Insecticide Cyhalothrin
J. Agric. Food Chem., Vol. 54, No. 15, 2006 5289
Table 4. Assay of Cross-Reactivity among Different Pyrhthroidsa
Table 6. Recovery of Cyhalothrin from the Fortified Water Samples
average recovery (%)
tap water well water
± SE (n ) 4)
spiked concn
g/L)
pretreatment
(
µ
wastewater
1
2
3
4
5
6
100
500
2500
100
500
2500
90.3
96.7
96.4
91.7
92.4
96.7
±
±
±
±
±
±
0.7
1.2
0.5
1.3
1.1
2.0
84.9
92.8
95.3
84.5
96.7
96.5
±
±
±
±
±
±
1.2
a
1.0
0.8
1.1
1.2
1.8
125
120
±
±
3.8
2.7
a
124
111
±
±
5.6
4.3
a
Because of lots of precipitate after adding methanol, the treatment could not
g/L);
be analyzed. Coating antigen (Cwb-OVA) concentration, 1/1000 (0.7
µ
antiserum (CWB-C) dilution, 1/4000; and goat anti-rabbit IgG-HRP dilution, 1/1000.
Figure 3. Relationship between the concentration of cyhalothrin fortified
into water samples and those determined by ELISA. Tap water (
(), well
water ( ), and wastewater ( ).
0
×
a
Cross-reactivity was calculated as (I50 of cyhalothrin/I50 of analyte)
× 100%;
(Table 6). The results indicated that simple dilution could
ni, <10% inhibition was measured at the highest concentration (10 mg/L). All analytes
were prepared in PBS with 50% methanol. Coating antigen (Cwb-OVA) concentra-
effectively minimize interferences as long as the concentrations
were above the LOD. Dankwardt et al. (25) reported that the
influence of humic substances in soil samples on ELISA was
eliminated by addition of BSA solution to the assay buffer.
Similar results were observed in wheat samples (26), in apple
sample using rabbit serum albumin (RSA) (19), and in industrial
water samples using BSA (20). In this study, bovine blood
fibrinogen, differing from the immunogens (BSA) and the
coating antigen (OVA) for no interference with the ELISA, was
chosen to reduce the interference from the sample impurities
with the ELISA. Without cleanup, well water had more ionic
impurities and other impurities than tap water. Pretreatment 5
(10-fold dilution and addition of bovine blood fibrinogen) for
well water had a better recovery (96.7%) than other pretreat-
ments. Wastewater had lots of salts and chemical impurities.
The permethrin immunoassay required 50 times dilution of
industrial water in order to obtain good analysis results (20).
The results of this study suggested that wastewater samples were
required to be diluted 50-fold (pretreatment 6) or higher and
bovine blood fibrinogen needed to be added for accurate analysis
of cyhalothrin in wastewater (Table 6). Figure 3 shows a good
correlation between the fortification levels of cyhalothrin in the
water samples and those determined recovery by the ELISA.
The average recoveries of cyhalothrin ranged from 90 to 120%
(Table 7). The results demonstrate that these pretreatments and
assays are suitable for the detection of the trace levels of
cyhalothrin in tap water, well water, and wastewater. Comparing
SPE and pretreatment, the method of SPE was more time-
consuming than the method of pretreatment but it made the
ELISA detect cyhalothrin in water at sub-part per billion levels.
The method of pretreatment was simple and timesaving, but it
increased the detection lower limit of the ELISA for cyhalothrin
tion, 1/1000 (0.7
IgG-HRP dilution, 1/1000.
µg/L); antiserum (CWB-C) dilution, 1/4000; and goat anti-rabbit
Table 5. Comparison of SPE Cyhalothrin Recoveries Determined by
ELISA with Those by GCa
spiked concn
g/L)
0.1
average recovery (%) (n
)
4)
water type
tap water
(µ
ELISA GC
100
96
±
±
±
±
±
±
±
±
±
9
6
104
±
±
±
±
±
±
±
±
±
2.5
1
0
.5
96
96
5
98
5
1
well water
0.1
89
10
7
88
2
0
.5
88
104
81
91
85
95
100
85
84
84
3
3
1.5
2.5
2
5
4
wastewater
0.1
3
0.5
9
5
8
a
Coating antigen (Cwb-OVA) concentration, 1/1000 (0.7 µg/L); antiserum (CWB-
C) dilution, 1/4000; and goat anti-rabbit IgG-HRP dilution, 1/1000.
compounds and trapped by C18 column, the matrix effects of
water samples were largely eliminated by prior SPE. However,
considering a simple pretreatment without cleaning up, it is
necessary to test the matrix effect from a real water sample. In
real water sample analysis, interference in the assay has been
caused by sample characteristics, such as ionic strength, pH,
and organic impurity (24). To adjust the ionic composition and
pH value of the samples, the samples were diluted in 50%
methanol-PBS. The ionic concentration of tap water was low,
and the pH value was close to 7. There were little organic
chemicals in the tap water. There were not many differences
between the recoveries of the tap water pretreatments 2, 3, 5,
and 6, but they were higher than the pretreatments 1 and 4

5290 J. Agric. Food Chem., Vol. 54, No. 15, 2006
Gao et al.
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±
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6
8
4
8
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98
97
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a
Ten-fold dilution in PBS with 50% methanol. b Ten-fold dilution in PBS with
0% methanol and added 0.2% bovine blood fibrinogen in the PBS solution for
diluting antibody. Fifty-fold dilution in PBS with 50% methanol and added 0.2%
bovine blood fibrinogen in the PBS solution for diluting antibody. Coating antigen
Cwb-OVA) concentration, 1/1000 (0.7 µg/L); antiserum (CWB-C) dilution, 1/4000;
5
7
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(
and goat anti-rabbit IgG-HRP dilution, 1/1000.
(
in water. In this test, SPE and pretreatment supplemented each
other. Using the method of SPE, the ELISA could detect
cyhalothrin in water at sub-part per billion levels. Using the
method of pretreatment, the ELISA could detect cyhalothrin in
water at part per billion levels and part per million levels. The
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in rabbits. An ELISA procedure was optimized for the analysis
of cyhalothrin in various types of water samples. The I50 and
LOD were 37.2 and 4.7 µg/L, respectively, and the calibration
linear range was 5-500 µg/L. This assay showed recoveries
averaging 97% for cyhalothrin spiked in tap water, well water,
and wastewater samples. The matrix effect of different water
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(
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ABBREVIATION USED
4
1, 2011-2018.
BSA, bovine serum albumin; DAPEC, 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide; DCC, 1,3-dicyclohexylcarbodiim-
ide; DMF, N,N-dimethylformamide; ELISA, enzyme-linked
immunosorbent assay; GAR-HRP, goat anti-rabbit immunoglo-
bulin conjugated to horseradish peroxidase; GC, gas chroma-
tography; HPLC, high-performance liquid chromatography; I50,
the concentration analyte giving 50% inhibition; LOD, limit of
detection; NHS, N-hydroxysuccinimide; NMR, nuclear magnetic
resonance; OVA, ovalbumin; PBS, phosphate-buffered saline;
RSA, rabbit serum albumin; SPE, solid phase extraction; TLC,
thin-layer chromatography; TMB, tetramethylbenzidine.
(
16) Lee, N.; McAdam, D. P.; Skerritt, J. H. Development of
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(
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ACKNOWLEDGMENT
We thank Dr. Xinlin Yang and Dr. Shangzhong Liu of China
Agricultural University for their assistance on hapten synthesis
and Dr. Guomin Shan of Dow AgroSciences LLC for sugges-
tions on the hapten design.
2
69-277.
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