Development of an enzyme-linked immunosorbent assay for the insecticide thiamethoxam

Article abstract of DOI:10.1021/jf0210472

An enzyme-linked immunosorbent assay (ELISA) was developed for the neonicotinoid insecticide thiamethoxam, 3-(2-chlorothiazol-5-ylmethyl)-5-methyl-4-nitroimino-1,3,5-oxadiazinane. Three antisera were raised from rabbits immunized with the hapten - KLH conjugate. On the basis of the computational analysis of hapten candidates, the hapten with a spacer arm on the thiazolyl ring of thiamethoxam was synthesized to elicit thiamethoxam-specific antisera. The hapten was 3-[2-(2-carboxyethylthio)-5-ylmethyl]-5-methyl-4-nitroimino-1,3, 5-oxadiazinane. Antisera were characterized with indirect competitive ELISA. Cross-reactivity and effects of organic solvents, pH, and ionic strengths were evaluated. The antiserum was specific for thiamethoxam and tolerant of up to 5% acetonitrile and 5% acetone. Various ionic strengths and pH values in the tested ranges had negligible effect on the assay performance. Under the optimized conditions, the half-maximal inhibition concentration (IC₅₀) and the limit of detection were approximately 9.0 and 0.1 μg/L of thiamethoxam, respectively. ELISA analysis of stream and tap water samples showed an excellent correlation with the fortification levels.

Full text of DOI:10.1021/jf0210472

J. Agric. Food Chem. 2003, 51, 1823−1830 1823
Development of an Enzyme-Linked Immunosorbent Assay for
the Insecticide Thiamethoxam
HEE-JOO KIM, SHANGZHONG LIU, YOUNG-SOO KEUM, AND QING X. LI*
Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa,
Honolulu, Hawaii 96822
An enzyme-linked immunosorbent assay (ELISA) was developed for the neonicotinoid insecticide
thiamethoxam, 3-(2-chlorothiazol-5-ylmethyl)-5-methyl-4-nitroimino-1,3,5-oxadiazinane. Three antisera
were raised from rabbits immunized with the hapten-KLH conjugate. On the basis of the computational
analysis of hapten candidates, the hapten with a spacer arm on the thiazolyl ring of thiamethoxam
was synthesized to elicit thiamethoxam-specific antisera. The hapten was 3-[2-(2-carboxyethylthio)-
5-ylmethyl]-5-methyl-4-nitroimino-1,3,5-oxadiazinane. Antisera were characterized with indirect
competitive ELISA. Cross-reactivity and effects of organic solvents, pH, and ionic strengths were
evaluated. The antiserum was specific for thiamethoxam and tolerant of up to 5% acetonitrile and
5% acetone. Various ionic strengths and pH values in the tested ranges had negligible effect on the
assay performance. Under the optimized conditions, the half-maximal inhibition concentration (IC50)
and the limit of detection were approximately 9.0 and 0.1 µg/L of thiamethoxam, respectively. ELISA
analysis of stream and tap water samples showed an excellent correlation with the fortification levels.
KEYWORDS: ELISA; immunoassay; thiamethoxam; neonicotinoid insecticide; hapten design
INTRODUCTION
dures, and potential risks, are presented in refs 23-25. The
major concerns are wind drift and leaching of thiamethoxam
into surface water and ground water (23), toxicities to a few
vulnerable aquatic species such as mysid shrimp and rainbow
trout embryos (25), toxicity to honey bees, parasitic wasps, and
other beneficial insects (25), and the development of resistance
by target insects (24). In addition, thiamethoxam in the formu-
lation known as Actara25-WG may lose activity or become
phytotoxic when mixed with certain fungicides such as man-
cozeb, metalaxyl, and folpet (26). Despite these drawbacks,
thiamethoxam, imidacloprid, and other neonicotinoids are
expected to replace organophosphates as the most widely used
insecticides worldwide (23).
Thiamethoxam and related neonicotinoids also have low acute
dermal and inhalation toxicities and in normal use do not cause
allergic reactions or skin or eye irritation in humans and animals.
Because of their high affinity for insect nAChRs (9), neo-
nicotinoids are very potent, and application of 5-8 oz/acre is
effective for some soil and foliar applications.
Currently, two generations of neonicotinoid insecticides are
in use. These are the chlorothiazolyl derivatives, such as imi-
dacloprid, and the thianicotinyl derivatives, exemplified by thia-
methoxam. High-performance liquid chromatography (HPLC)
and HPLC-mass spectrometry (HPLC-MS) are currently pre-
ferred for determination of neonicotinoids in environmental
samples (23). The thermolability and high polarity of neonico-
tinoids make them difficult to analyze with gas chromatography
(GC) or GC-MS (27-29). However, the water solubility, asym-
metric structure, and potential for leaching into surface water
and ground water make ELISA potentially a good analytical
Thiamethoxam, 3-(2-chlorothiazol-5-ylmethyl)-5-methyl-4-
nitroimino-1,3,5-oxadiazinane, belongs to a relatively new class
of insecticides known as neonicotinoids. Thiamethoxam, imi-
dacloprid, and related neonicotinoids have several advantages
over pyrethroid, organophosphate, and carbamate insecticides
and are rapidly replacing them worldwide.
Pyrethroids interact mainly with presynaptic sodium channels
in vertebrates as well as insects (1-3). Organophosphate and
carbamates act by inhibiting acetylcholine esterases and thus
are toxic to vertebrates, insects, and other invertebrates (4). By
contrast, neonicotinoids act similarly to nicotinic acid as agonists
of the postsynaptic nicotinic acetylcholine receptors (nAChR).
Neonicotinoids are 100-fold or more selective for insect nAChRs
than for vertebrate nAChRs (5, 6). Several research groups
worldwide have published evidence related to the submolecular
basis for this selectivity, from the standpoint of the receptor
subunit composition and properties, as well as the steric and
charge distribution characteristics of the neonicotinoids (6-18).
The U.S. EPA promulgated rules for thiamethoxam use
beginning in 2000 (19-22). During 2001, thiamethoxam was
approved for various uses by the Massachusetts Department of
Food and Agriculture, the Canadian Pest Management Regula-
tory Agency, and the Australian National Registration Authority
for Agricultural and Veterinary Chemicals. Abundant details
of the chemistry, metabolism, bioavailability, human toxicology,
and ecotoxicology, as well as approved uses, analytical proce-
* Author to whom correspondence should be addressed [telephone (808)
956-2011; fax (808) 956-8384; e-mail qingl@hawaii.edu].
10.1021/jf0210472 CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/27/2003

1824 J. Agric. Food Chem., Vol. 51, No. 7, 2003
Kim et al.
method. To date, our laboratory and one other have developed
haptens and ELISAs based on rabbit sera, and Watanabe et al.
derived another hapten and monoclonal mouse antibodies for
ELISA of the most heavily used chlorothiazolyl neonicotinoid
insecticides, imidacloprid and acetamiprid (30-32). In this paper
we describe the design of a hapten and development of serum-
based direct and indirect competition ELISAs for thiamethoxam,
the most used thianicotinyl insecticide in this class. Hapten
design was facilitated by computational modeling of the energy-
minimized structures and their charge distributions.
MATERIALS AND METHODS
Reagents. All reagents were of analytical grade unless specified
otherwise. Reference standards of clothianidin (99.9%), acetamiprid
(99.5%), and dinotefuran (99.7%) were kindly provided by the National
Institute of Agricultural Science and Technology, South Korea. Primary
stock solutions were prepared by dissolving each reference standard in
methanol at a concentration of 0.5 mg/mL. Chemicals purchased from
Sigma (St. Louis, MO) were goat anti-rabbit IgG-horseradish peroxi-
dase (IgG-HRP), keyhole limpet hemocyanin (KLH), bovine serum
albumin (BSA), HRP, phosphate-citrate buffer capsules with sodium
perborate, carbonate-bicarbonate buffer capsules, and o-phenylenedi-
amine (OPD). TiterMax Gold adjuvant was obtained from CytRx Corp.
(Norcross, GA). The ELISAs were carried out in 96-well polystyrene
microplates (MaxiSorp F96, Nalge Nunc International, Roskilde,
Denmark). Thiamethoxam, the thiamethoxam hapten, and the hapten
conjugates were synthesized in this laboratory. The antiserum was
purified with a HiTrap Protein G HP column (Amersham Pharmacia
Biotech, Piscataway, NJ) according to the manufacturer’s instructions.
Concentration of antibody in the final preparation was determined with
the Bio-Rad Bradford protein assay (Bio-Rad Laboratories, Hercules,
CA). The purified IgG in phosphate-buffered saline (PBS, 12 mM
phosphate, 137 mM NaCl, and 2.7 mM KCl, pH 7.5) was stored at
-20 °C.
Spectroscopy and Chromatography. 1H NMR spectra were
obtained on a Nicolet NT-300 MHz for solutions in CDCl3 and are
described as multiplicity, coupling constant (J) in hertz (Hz), number
of protons, and assignment. Chemical shifts (δ, ppm) are relative to
internal tetramethylsilane (TMS). Low-resolution mass spectra were
recorded with a Hewlett-Packard 5958 GC-MS system with an electron
impact (EI, 70 eV) ionization and are given as [M]+.
Molecular Modeling. Global energy minimum structures were
searched by preliminary conformational analysis with all of the rotatable
bonds, and refined energy minimization was done by PM5 force field.
To evaluate the structural similarity, root-mean-square (RMS) deviations
were calculated by superimposing other chemicals on thiamethoxam.
Atomic properties, including superdelocalizability, partial charge, and
electron density, were calculated with the same force field. Electron
density isosurfaces colored by electrostatic potential were also calculated
to evaluate the electron distribution over the molecules. All of the
calculations were done by CAChe Worksystem Pro (version 5.0, Fujitsu,
Japan). Partial dipole moment, molecular dimension, and hydrogen
bonding properties were calculated with Molecular Modeling Pro
(version 5.0.8, ChemSW Inc.). Atomic polarizability and partial π and
σ charges were calculated with PETRA (33).
Safety Precaution. Hapten syntheses were done in a chemical fume
hood with charcoal filters. All synthetic byproducts and wastes and
solutions of thiamethoxam analytes were disposed of as hazardous
materials. Goggles, spill-resistant gowns, and chemical-impermeable
nitrile gloves were worn.
Synthesis of Hapten (Figure 1). In general, the final hapten,
thiamethoxam, and intermediates were synthesized according to
established methods (30, 34-37).
Preparation of 3-Methyl-4-nitroimino-1,3,5-oxadiazinane (I). A
solution of N-methyl-N′-nitroguanidinene (6.0 g) and 37% aqueous
formaldehyde solution (37.5 mL) was added to 37.5 mL of 90% formic
acid and heated to 80 °C for 16 h. After the solution had cooled to 0
°C, an aqueous sodium hydroxide solution (300 g/L, ∼70 mL) was
added to adjust the solution to pH 8.0. The resulting mixture was
Figure 1. Synthetic scheme of thiamethoxam hapten.
extracted with CH2Cl2. The organic phase was washed with saturated
sodium chloride, dried by anhydrous sodium sulfate, and evaporated
to dryness. The solid residue was recrystallized from ethyl acetate and
ethyl ether to get a white solid: 5.5 g, yield 67%; mp 141-143 °C;
GC-MS C4H8N4O3, 160 (M+).
Preparation of 2-Chloro-5-methylthiazole (II). A solution of 2-amino-
5-methylthiazole (10 g) in 40 mL of concentrated HCl and 20 mL of
water was cooled to 0 °C. Aqueous sodium nitrite (7 g in 15 mL of
water) was added dropwise to the reaction mixture. After the mixture
had been maintained at 0 °C for 3 h, the solution was heated to 80 °C
for 3 h and then cooled to room temperature. The reaction mixture
was extracted with chloroform (400 mL × 3), and the extracts were
combined and dried with anhydrous sodium sulfate. The solvent was
removed under reduced pressure, and the residue was purified with a
silica gel column and eluted with CH2Cl2: GC-MS C4H4ClNSH+, 134
(MH+).
Preparation of 2-Chloro-5-bromomethylthiazole (III). In a 50-mL
round-bottom flask, a solution of II (8.0 g), N-bromosuccinimide (NBS)
(12.8 g), and benzoyl peroxide (AIBN) (28.8 mg) in CCl4 (200 mL)
was heated to 80 °C for 36 h, cooled to room temperature, and filtered.
The filtrate was washed to neutral with water, 5% sodium bicarbonate,
and water and dried by anhydrous sodium sulfate. After solvent removal,
the residue was purified by flash chromatography (ethyl acetate/hexane
) 1:9): yield 85%; GC-MS C4H3BrClNSH+, 213 (MH+).
Preparation of Thiamethoxam (IV). A solution of I (3.0 g), III (5.0
g), and potassium carbonate (6.25 g) in DMF (20 mL) was heated to
30 °C overnight and then to 50 °C for 16 h. After the reaction mixture
had been filtered through Celite, DMF was removed at reduced pressure.
The residue was purified by flash chromatography (CH2Cl2/CH3OH )
95:5). The purified product has mp 140-142 °C; LC-MS C8H10ClO3-
1
SH+, 292 (MH+); H NMR (DMSO-d6) δ 2.832 (3H, s, CH3), 4.746

ELISA for Thiamethoxam
J. Agric. Food Chem., Vol. 51, No. 7, 2003 1825
(2H, s, CH2), 4.968 (2H, s, CH2), 5.039 (2H, s, CH2), and 7.639 (1H,
s, CHd).
Preparation of 3-[2-(2-Carboxyethylthio)-5-ylmethyl]-5-methyl-4-
nitroimino-1,3,5-oxadiazinane (V). In a 50-mL round-bottom flask, a
solution of thiamethoxam (0.5 g), 3-mercaptopropionic acid (0.2 g),
and 85% potassium hydroxide (powder, 0.2 g) in DMSO (15 mL) was
heated to 75 °C for 3 days. The solution was poured into water (100
mL), adjusted to pH 3 with 1 N HCl, and then extracted with ethyl
acetate (30 mL × 3). The organic phase was combined, washed with
brine solution, and dried with anhydrous Na2SO4. After the solvent
was removed at reduced pressure, the residue was dissolved in methanol.
The product was separated by preparative thin-layer chromatography
(CH2Cl2/CH3OH ) 9:1). The yield was 55.6%: LC-MS C11H15N5O5S2H+,
362 (MH+); 1H NMR (DMSO-d6) δ 2.756 (2H, t, CH2, J ) 7.0
Hz), 2.980 (3H, s, CH3), 3.406 (2H, t, CH2, J ) 7.0 Hz), 4.806
(2H, s, CH2), 4.976 (2H, s, CH2), 5.032 (2H, s, CH2), and 7.616 (1H,
s, CHd).
Preparation of Protein)Hapten Conjugates. Hapten-KLH and
-BSA Conjugation. The hapten (V) was conjugated to KLH and BSA
as previously described (30) with slight modification. To 0.05 mmol
of the hapten was added 500 µL of DMF solution containing 0.1 mmol
each of N-hydroxysuccinimide (NHS) and 1-[3-(Dimethylamino)pro-
pyl]-3-ethylcarbodimide hydrochloride (DEC). The mixture was stirred
at room temperature for 4 h and then centrifuged to remove precipitated
urea. The clear supernatant was slowly added, a few microliters per 20
min intervals, to KLH solution (30 mg of KLH in 10 mL of a 0.1 M
borate buffer, pH 9.0) or BSA solution (150 mg of BSA in 10 mL of
a 0.1 M borate buffer, pH 9.0). The reaction mixture was stirred
overnight at 4 °C and then dialyzed against PBS (1.5 L) for 3 days at
4 °C with two buffer changes per day.
Preparation of Enzyme Tracer. The hapten-HRP conjugate was
prepared as above with the following exception. Hapten (6 µmol) was
activated with NHS (30 µmol) and DEC (60 µmol) in 260 µL of DMF,
and the reaction mixture was added to HRP (2 mg) in 3 mL of 0.13 M
NaHCO3 buffer. After the dialysis against PBS, the crude hapten-
HRP conjugate was diluted with an equal volume of glycerol and stored
at -20 °C.
Immunization. The hapten-KLH conjugate was used to immunize
rabbits. A biochemically defined adjuvant, TiterMax Gold, was selected
due to its ability to produce high titers of antisera (38). Three New
Zealand white rabbits (2-4 kg) were immunized with hapten-KLH
emulsified in TiterMax Gold as previously described (30). One received
60 µg and the others received 100 µg of emulsion in 0.4 mL. Two
booster injections were given at 4-week intervals. Injections were made
intradermally and subcutaneously at multiple sites on the animals’ backs.
The titers of the antisera were monitored by ELISA using checkerboard
titration. Seven days after the last injection, the rabbits were bled, and
the antisera were collected and stored at -80 °C. Inhibition of antibody
binding by free thiamethoxam was monitored with an indirect competi-
tive ELISA (icELISA) (30).
Figure 2. Structures and dihedral angles of thiamethoxam and hapten
candidates.
determine thiamethoxam concentrations in the fortified water samples.
The samples were buffered with 4-fold concentrated PBST.
RESULTS AND DISCUSSION
Hapten Design and Synthesis. There are several possible
linker positions (X, Y, and Z) in thiamethoxam for hapten
preparation (Figure 2). To obtain compound-specific antibodies,
a good option is to place a linker on a remote part of the mole-
cule and to maximally reserve the parent molecular property
(39, 40). The hapten design was aided with computational analy-
sis of hapten candidates. Various chemical and physical proper-
ties of thiamethoxam and its analogues were predicted with
computational modeling to identify a suitable linker position
(Figures 2 and 3; Table 1). The computational analysis sug-
gested that a thioalkyl spacer on the 2-position of the thiazolyl
ring alters the molecular properties much less than one on the
4-position of the thiazolyl ring or the 5-position of the oxadi-
azinane ring (Figures 2 and 3; Table 1). In addition, replacing
the N-methyl group with a spacer (proposed hapten C) may
not produce a suitable hapten because clothianidin and thia-
methoxam share the same thiazole structure, located at the end
of the molecule remote from the spacer. The proposed hapten
B may not be an adequate hapten either because a large steric
hindrance of the linker at the 4-position of thiazole significantly
distorts the geometry from that of thiamethoxam (Figure 3;
Table 1). These considerations suggest that the proposed haptens
B and C would not elicit thiamethoxam-specific antibodies. The
computational modeling indicated that hapten A has almost the
same geometric overlap and electrostatic properties as thiameth-
oxam (Figure 3; Table 1) and is a suitable hapten candidate.
Therefore, it was synthesized and used to induce antibodies.
Thiamethoxam (IV) and three intermediates (I-III, Figure
1) were synthesized according to established methods (34-37).
Thiamethoxam was prepared by reaction of compounds I and
III as shown in Figure 1. Introduction of the spacer to the
selected site of thiamethoxam was carried out under a mild
alkaline condition because conversion of dNsNO2 to dO at a
strong alkaline condition was observed in the previous work
(30). The molecular ratio of hapten to protein was estimated
by UV absorbance and was 10 and 43 for hapten-BSA and
hapten-KLH, respectively.
Direct Competitive ELISA (dcELISA). A microtiter plate was
coated with rabbit anti-thiamethoxam-KLH antibody (100 µL of 2.0
µg antibody/well in 0.1 M carbonate-bicarbonate buffer, pH 9.6)
overnight at 4 °C. The following day, the plate was washed four times
with PBS containing 0.05% Tween 20 (PBST) and then blocked with
1% BSA in PBS (200 µL/well) for 1 h at room temperature. After the
plate was washed (five times) with PBST, a solution of 50 µL of the
analytes or standards diluted in PBST and 50 µL of thiamethoxam-
HRP conjugate (0.16 ng/µL in PBST) was added. After 40 min of
incubation at 37 °C and another five washings, the substrate solution
(100 µL per well, containing 1.0 mg/mL of OPD in 0.05 M citrate-
phosphate and 0.03% sodium perborate, pH 5.0) was added. The
reaction was stopped with sulfuric acid (4 N, 50 µL/well) after 15 min
at room temperature, and absorbance at 490 nm was read with a Vmax
microplate reader (Molecular Devices, Sunnyvale, CA). Inhibition
curves were fit to a four-parameter logistic equation with Softmax
software v 2.35 (Molecular Devices).
Immunization and Characterization of Antisera. Table 2
shows the titers and IC50 values of the three antisera for free
thiamethoxam. The titer of antiserum-II (Ab-II) from rabbit
II was 5- and 10-fold higher than those from rabbits I (Ab-I)
and III (Ab-III), respectively. However, the antiserum-I
(Ab-I) from rabbit I had the lowest IC50 (278 ng/mL) in an
icELISA (Table 2). Protein G affinity-purified Ab-I was stored
at -20 °C and used throughout this work. When compared with
the titers of imidacloprid antisera obtained with the same proce-
dure (30), the titers of these thiamethoxam antisera were low,
which may be due to the lower ratio of thiamethoxam hapten
to KLH and differences in the hapten structures and individual
animals.
Fortification of Thiamethoxam in Water. Water samples were
from a tap in the laboratory and were collected from Manoa stream in
Honolulu, HI. Tap and stream water samples were fortified with
thiamethoxam up to 100 ng/mL. The optimized dcELISA was used to

1826 J. Agric. Food Chem., Vol. 51, No. 7, 2003
Kim et al.
Figure 3. Electron density isosurfaces colored by electrostatic potential: white, >0.09; 0.03 < red < 0.09; 0.01 < yellow < 0.03; 0.00 < pale green < 0.01;
−0.01 < pale blue < 0.00; −0.03 < blue < −0.01; −0.06 < pink < −0.03; gray < −0.06. The structures of haptens B and C were proposed but not
synthesized.
Table 1. RMS Errors of the Superimposition on Thiamethoxam and Molecular Dimensions of Global Energy Minimum Structures
dihedral angle (deg)
A2
molecular dimension (Å)
width
compound
acetamiprid
clothianidin
dinotefuran R
dinotefuran S
imidacloprid
thiamethoxam
RMS deviation
A1
A3
length
depth
0.1092
0.0982
0.2813
0.2796
0.1127
0.0000
0.0242
0.2868
0.0562
−1.2
−61.8
−3.2
−3.4
−0.2
−119.3
−119.7
−89.1
−105.2
70.1
74.3
−169.9
115.2
69.0
82.9
82.3
155.2
84.4
−111.1
−125.8
173.2
−71.7
−90.0
−72.9
−73.9
−79.8
−73.8
11.35
10.80
11.20
11.10
10.68
12.17
12.16
12.35
12.15
8.77
8.78
8.82
8.82
9.18
9.01
9.03
9.12
9.11
7.62
7.67
6.16
6.35
7.98
7.91
7.92
7.82
7.53
hapten A (synthesized)
hapten B (proposed)
hapten C (proposed)
Competitive Inhibition. Figure 4 shows representative
standard curves for thiamethoxam obtained with dcELISA and
icELISA. The calibration ranges of the dcELISA and icELISA
curves were approximately 0.1-200 and 10-5000 µg/L,
respectively. The IC50 values of dcELISA and icELISA were
approximately 8 and 160 µg/L, respectively, indicating that use
of dcELISA and purification of antibody improved the sensitiv-
ity by 20-fold compared with that of an icELISA. A similar
improvement with the dcELISA format was observed with the
imidacloprid polyclonal antibody (40). A dcELISA format using

ELISA for Thiamethoxam
J. Agric. Food Chem., Vol. 51, No. 7, 2003 1827
volume of the pyridine of acetamiprid and imidacloprid (55.29
cm2/mol) is similar to that of the thiazole of thiamethoxam and
clothianidin (Figure 2). In addition, there appeared to be similar
electrostatic properties between the pyridine and thiazole rings.
Large IC50 differences for thiamethoxam, clothianidin, and
imidacloprid indicate that the conservation of the nitroimi-
nothiadiazinane moiety in the thiamethoxam hapten distal to
the linker was important for the production of thiamethoxam-
specific antibodies. Clothianidin and thiamethoxam have a
chlorothiazole moiety; however, the large structural differences
in their nitroimino groups probably result in low binding affinity
of clothianidin to the antibody. The electrostatic properties of
imidacloprid are more similar to thiamethoxam than clothianidin,
but large differences exist in dihedral angle A1 among imida-
cloprid, clothianidin, and thiamethoxam (Table 1). The large
IC50 value for imidacloprid strongly suggests that the geom-
etry of the nitroimino group is an important factor for the
selectivity between chemicals (Figure 3; Table 1). The R- and
S-enantiomers of dinotefuran share the same nitroimino structure
with clothianidin, but dihedral angle A1 and the molecular
shapes are completely different. Acetamiprid shows good
geometric overlap with thiamethoxam, but the large electrostatic
difference permits no cross-reactivity.
Table 2. Titer and Apparent Binding Affinity of Antisera
antiserum
titera
IC b (ng/mL)
50
I
II
III
8000
40000
4000
280
>1500
>1200
a The titer of antiserum is defined as the antiserum dilution that produced an
absorbance of 1 at 490 nm, 20 min after the addition of the substrate. b IC50 values
represent the concentration of thiamethoxam in ng/mL that produced 50% inhibition
of antiserum binding to the hapten conjugate using icELISA. The wells were coated
with 100 µL of conjugate of 25 ng/mL.
Chemical Effects on Assay Performance. Assay perfor-
mance is often affected by ionic strength, pH, and solvent
concentration of samples. The pH of aqueous samples may
interfere with ELISAs due to the disturbance of weak mo-
lecular interactions in antibody-antigen binding caused by pH
derived alteration (e.g., hydrolysis, conformational change,
ionization) of either an analyte or molecules (antibody or en-
zyme tracer) participating in the interaction (42-44). Salt
concentrations in real world samples can often affect assays
(40, 44). The effects of these parameters on the assay were
evaluated.
Figure 4. Standard inhibition curves of thiamethoxam in dcELISA and
icELISA formats. Plates were coated with 100 µL of 2.0 µg of purified
Ab-I for dcELISA or 2.5 ng of hapten−BSA per well for icELISA. Each
value represents the mean of four replicates.
Table 3. Cross-Reactivity of Ab-I to Thiamethoxam and Its Structural
Analoguesa
SolVent Effects. The effects of DMSO, MeOH, acetone, and
acetonitrile were studied because they are water-miscible and
are commonly used in sample extractions. Serial dilutions of
thiamethoxam standard in PBST containing 0, 5, 10, 20, or 40%
of organic solvent were mixed with an equal volume of antibody
solution diluted in PBST for dcELISA. In general, maximum
absorbance (Amax) gradually decreased as the concentration of
solvent increased. Acetonitrile and acetone showed less effect
on the inhibition curves (shape and position) than MeOH and
DMSO (Figure 5). Little change in IC50 values was observed
in up to 5% of acetonitrile and 5% of acetone. However, IC50
values increased gradually as concentrations of MeOH and
DMSO increased.
compound
IC50 (nM, ng/mL)
cross-reactivity (%)
thiamethoxam
thiamethoxam hapten
clothianidin
29.3 (9.0)
14.8 (10)
1587 (420)
100
198
1.8
imidacloprid
3462 (885)
0.8
dinotefuran
acetamiprid
>11 510 (>2500)
>6740 (>1500)
<0.25
<0.4
a The plate was coated with 2.0 µg of affinity-purified antibody (100 µL/well).
b Numbers in parentheses are IC50 values in ng/mL.
the purified Ab-I was employed throughout this study because
it significantly improved the assay sensitivity.
Effect of pH and Ionic Strength. To evaluate the influence of
pH and ionic strength on assay performance, thiamethoxam was
diluted in PBST of different pH values (4.0-9.0) with the same
ionic strength or different ionic strengths (0.15-1.5 M) at pH
7.5 and enzyme tracer in PBST, pH 7.5. Amax values decreased
at pH <6; however, there were negligible shifts of the inhibition
curves in the pH range of 4-9 (data not shown). All subsequent
assays were performed at pH 7.0. There were no significant
changes in IC50 values and Amax in the tested range of ionic
strength (data not shown).
Cross-Reactivity. The specificity of Ab-I was estimated by
performing dcELISA with four neonicotinoid insecticides as
competitors, which were imidacloprid, acetamiprid, dinotefuran,
and clothianidin. Ab-I was specific for thiamethoxam and
showed <2% cross-reactivity for imidacloprid, chlothianidin,
and acetamiprid (Table 3). No response in the assay was
observed up to 12 µM dinotefuran. These results support the
hapten design suggested by the computational analysis.
The relationship between the antibody specificity and com-
petitor structures was examined by computational modeling. The
largest structural difference was found in dihedral angles (Figure
2; Table 1). The nitro group in thiamethoxam appears to be
distorted into a nearly perpendicular position (dihedral angle
A1) to the thiadiazinane ring, which coincides well with
crystallographic data of structurally related N-methylimidaclo-
prid (41). Computational modeling suggested that the steric
Thiamethoxam-Fortified Water Samples. Thiamethoxam
in water samples was analyzed with the dcELISA. The
concentrations of thiamethoxam by ELISA correlated very well
with the fortification values with a slope of 0.94 and a
correlation coefficient of 0.99 for the tap water samples and a
slope of 0.94 and a correlation coefficient of 0.99 for the

1828 J. Agric. Food Chem., Vol. 51, No. 7, 2003
Kim et al.
Figure 6. Correlation between thiamethoxam concentrations determined
by ELISA and those fortified in tap water and stream water samples. The
plate was coated with 2.0 µg of Ab-I in 100 µL/well. The concentration
of enzyme tracer was 0.16 ng/µL in PBST, pH 7.0. Each value represents
the mean of four replicates.
stream water (Figure 6). The results showed that the ELISA
can accurately measure the concentration of thiamethoxam in
water.
Summary and Conclusions. Thiamethoxam-specific poly-
clonal antibodies were obtained from rabbits immunized with
a hapten-KLH conjugate of hapten having a thioalkyl spacer
at the thiazolyl ring of thiamethoxam. The hapten design was
aided by computational analysis of RMS deviations, dihedral
angles, and molecular dimensions of candidate haptens. The
experimental results were consistent with our prediction that
the substitution of the chlorine atom with a thioalkyl group is
a suitable position to derivatize in as well to obtain thia-
methoxam-specific antibodies. All of the molecules that have a
flat dihedral angle (A1) showed little cross-reactivity. In
addition, the nitro group configuration and the presence of the
oxadiazinane group may play a key role in the antigen-antibody
recognition.
An ELISA was developed for the detection of thiamethoxam.
The use of the protein G-purified IgG and the dcELISA format
significantly improved sensitivity and analysis time for the
thiamethoxam ELISA. The assay was specific for thiamethoxam
with an IC50 of 29 nM and had <2% cross-reactivity with
structural analogues of thiamethoxam. The good recovery of
thiamethoxam in water samples showed that the assay can be
used for the determination of thiamethoxam residues in water,
but further work is needed to validate this assay with other
matrices.
ACKNOWLEDGMENT
We thank Dr. Sylvia Kondo for assistance with rabbit im-
munization and Beth Irikura and Dr. Lassie Tam for review of
the manuscript.
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