New approach to immunochemical determinations for triclopyr and 3,5,6-trichloro-2-pyridinol by using a bifunctional hapten, and evaluation of polyclonal antiserum.

Article abstract of DOI:10.1021/jf0114684

The present work describes the design and synthesis of the structurally unique hapten, "bifunctional hapten", to produce a group-specific polyclonal antiserum to triclopyr and 3,5,6-trichloro-2-pyridinol. A bifunctional hapten was designed and synthesized by conjugating commercially available Nepsilon-2,4-dinitrophenyl (DNP)-L-lysine to triclopyr, and then coupling this to carrier proteins such as bovine serum albumin (BSA). The synthesized bifunctional hapten greatly raised the antiserum titer in comparison with that of the conventional hapten, triclopyr. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 63 days after the primary immunization. The obtained antiserum showed the highest affinity to triclopyr (IC(50) = 3.5 nM) and 3,5,6-trichloro-2-pyridinol (IC(50) = 5.1 nM) in homologous ELISA. The cross-reactivities to various agrochemicals and some chlorinated phenolic compounds were determined. Significant cross-reactivity was found to the herbicide 2,4,5-T. The antiserum reacted to both triclopyr and its metabolite. Assay sensitivity was evaluated for effects of various assay conditions, including pH value and concentrations of organic solvents and detergents. Under optimized assay conditions, the quantitative working range of triclopyr ELISA was from 0.1 to 5.2 ng/mL with a limit of detection (LOD) of 0.037 ng/mL, and an IC(50) of 0.72 ng/mL. On the other hand, the quantitative working range of 3,5,6-trichloro-2-pyridinol ELISA was from 0.13 to 6.0 ng/mL with a LOD of 0.052 ng/mL, and an IC(50) of 0.95 ng/mL. Water samples fortified with triclopyr or its metabolite at 1, 5, and 10 ng/mL were directly analyzed without extraction and cleanup by the proposed ELISA. The mean recovery was 101.6%, and the mean coefficient of variation (CV) was 7.1% in the case of the triclopyr ELISA. In the case of the 3,5,6-trichloro-2-pyridinol ELISA, the mean recovery was 99.8%, and the mean CV was 9.5%. The proposed ELISA turned out to be a powerful tool for monitoring of residual triclopyr or 3,5,6-trichloro-2-pyridinol in water samples at trace level.

Full text of DOI:10.1021/jf0114684

J. Agric. Food Chem. 2002, 50, 3637−3646 3637
New Approach to Immunochemical Determinations for Triclopyr
and 3,5,6-Trichloro-2-pyridinol by Using a Bifunctional Hapten,
and Evaluation of Polyclonal Antiserum
EIKI WATANABE,† RYOKO HOSHINO,† YUKIKO KANZAKI,† HIROSHI TOKUMOTO,†
HIROAKI KUBO,‡ AND HIROYUKI NAKAZAWA*,†
Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hoshi University,
2-4-41, Ebara, Shinagawa-ku, Tokyo 142-8501, Japan, and Department of Analytical Chemistry,
School of Pharmaceutical Sciences, Kitasato University, 5-9-1, Shirokane, Minato-ku,
Tokyo 108-8641, Japan
The present work describes the design and synthesis of the structurally unique hapten, “bifunctional
hapten”, to produce a group-specific polyclonal antiserum to triclopyr and 3,5,6-trichloro-2-pyridinol.
A bifunctional hapten was designed and synthesized by conjugating commercially available Nꢀ-2,4-
dinitrophenyl (DNP)-L-lysine to triclopyr, and then coupling this to carrier proteins such as bovine
serum albumin (BSA). The synthesized bifunctional hapten greatly raised the antiserum titer in
comparison with that of the conventional hapten, triclopyr. Antiserum with a sufficiently high titer to
provide the determinations of targeted compounds was obtained only 63 days after the primary
immunization. The obtained antiserum showed the highest affinity to triclopyr (IC50 ) 3.5 nM) and
3,5,6-trichloro-2-pyridinol (IC50 ) 5.1 nM) in homologous ELISA. The cross-reactivities to various
agrochemicals and some chlorinated phenolic compounds were determined. Significant cross-reactivity
was found to the herbicide 2,4,5-T. The antiserum reacted to both triclopyr and its metabolite. Assay
sensitivity was evaluated for effects of various assay conditions, including pH value and concentrations
of organic solvents and detergents. Under optimized assay conditions, the quantitative working range
of triclopyr ELISA was from 0.1 to 5.2 ng/mL with a limit of detection (LOD) of 0.037 ng/mL, and an
IC50 of 0.72 ng/mL. On the other hand, the quantitative working range of 3,5,6-trichloro-2-pyridinol
ELISA was from 0.13 to 6.0 ng/mL with a LOD of 0.052 ng/mL, and an IC50 of 0.95 ng/mL. Water
samples fortified with triclopyr or its metabolite at 1, 5, and 10 ng/mL were directly analyzed without
extraction and cleanup by the proposed ELISA. The mean recovery was 101.6%, and the mean
coefficient of variation (CV) was 7.1% in the case of the triclopyr ELISA. In the case of the 3,5,6-
trichloro-2-pyridinol ELISA, the mean recovery was 99.8%, and the mean CV was 9.5%. The proposed
ELISA turned out to be a powerful tool for monitoring of residual triclopyr or 3,5,6-trichloro-2-pyridinol
in water samples at trace level.
KEYWORDS: Triclopyr; 3,5,6-trichloro-2-pyridinol; bifunctional hapten; ELISA; cross-reactivity; water
analysis
INTRODUCTION
(1) and Woodburn et al. (2) reported that these compounds are
quickly transformed to triclopyr though photolysis and hydroly-
sis. The environmental dissipation and transformation of tri-
clopyr in river water and lake water was extensively studied by
Woodburn et al. (2) and Solomon et al. (3). Norris et al. (4)
reported that triclopyr undergoes biodegradation in aerobic soil
environments. The major degradation product is 3,5,6-trichloro-
2-pyridinol (Figure 1). Lickly and Murphy (5) reported that
3,5,6-trichloro-2-pyridinol is also the major metabolite produced
by fish. 3,5,6-Trichloro-2-pyridinol is also the major degradation
product of the organophosphorus insecticide chlorpyrifos and
chlorpyrifos-methyl. Chlorpyrifos is a broad-spectrum insecti-
cide that is widely used in agriculture and indoor disinfestation
The herbicide triclopyr (3,5,6-trichloro-2-pyridyloxyacetic
acid, Figure 1) has been manufactured and marketed by
DowElanco since the mid 1970s. Triclopyr can be applied as
either the triethylamine salt or the ethylene glycol butyl ether
ester, and is often used in mixed formulation with other
phenoxyalkanoic acids such as 2,4-D, mecoprop, and so on for
control of woody plants and many broad-leaved weeds in
grasslands, uncultivated lands, and rice fields. McCall and Gavit
* To whom correspondence should be addressed [telephone +81-3-5498-
5763; fax +81-3-5498-5062; E-mail nakazawa@hoshi.ac.jp].
† Hoshi University.
‡ Kitasato University.
10.1021/jf0114684 CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/16/2002

3638 J. Agric. Food Chem., Vol. 50, No. 13, 2002
Watanabe et al.
for pesticide residues in environmental samples (27-29) and
food samples (30-32). We developed a new type of hapten,
“bifunctional hapten”, with two functions: the conventional
function of producing an antibody against an antigen, and a
unique function of accelerating the production of the antibodies
in the animal (unpublished data). Based on this unique concept,
we succeeded in getting a group-specific polyclonal antiserum
to the organophosphorus insecticide fenitrothion, its major
metabolite product fenitrooxon, and its major degradation
product 3-methyl-4-nitrophenol in 40 days only [unpublished
data]. Although several immunoassays have been reported for
the detection of triclopyr (29) and 3,5,6-trichloro-2-pyridinol
(33), we designed the bifunctional hapten to obtain polyclonal
antisera to these compounds based on the concept in the present
study. The effect of synthesized bifunctional hapten was
evaluated by comparison with conventional hapten, and the
characterization of the obtained polyclonal antiserum for
sensitivity and specificity was presented. Furthermore, the
influence of the assay’s performance in water matrix was
evaluated.
Figure 1. Chemical structures of triclopyr, 3,5,6-trichloro-2-pyridinol, and
synthesized hapten (LysTCPY). Triclopyr (TCPY) was also used as coating
hapten for indirect ELISA (checkerboard titration) and as immunogen for
production of antiserum R-21. Bifunctional hapten, LysTCPY, was used
as immunogen for production of antiserum R-20 and as optimal coating
hapten (homologous hapten) for triclopyr and 3,5,6-trichloro-2-pyridinol
ELISAs.
(6). Because of its widespread use in agriculture, a high
chlorpyrifos residue occurrence in food has been reported (7),
which poses potential health hazards (8). Furthermore, it was
found not only in food but also in water - surface water,
groundwater, or both (9, 10). Therefore, there is actually a
growing concern about toxicological and environmental risks
associated with the remaining chlorpyrifos residues after its
application. Because 3,5,6-trichloro-2-pyridinol is rapidly ex-
creted in human urine after exposure to chlorpyrifos, it is used
as a biomarker of exposure (11). On the basis of the above-
mentioned background, in the United States active surveys of
exposure to various pesticides were carried out (12, 13). Kutz
et al. (12) reported that 3,5,6-trichloro-2-pyridinol was found
in 5.8% of urine samples provided from the U.S. population,
and suggested that the results were consistent with the high
occurrence of the parent pesticides. On the other hand, Hill et
al. (13) also reported an interesting survey in which they
analyzed 12 compounds containing 3,5,6-trichloro-2-pyridinol
in urine samples of about 1,000 adults living in the U.S., and
found 3,5,6-trichloro-2-pyridinol in 82% of those at observed
maximum concentration of 77 ng/mL. They suggested that
exposure to chlorpyrifos appears to be increasing.
Current triclopyr analysis is mainly carried out by using gas
chromatography (GC) equipped with an electron capture detector
(ECD) (3, 14, 15). These analytical methods involve methylation
with diazomethane, which is an explosion hazard and requires
handling of highly carcinogenic precursors. On the other hand,
3,5,6-trichloro-2-pyridinol analysis is also mainly carried out
by instrumental analysis such as GC (16-19) or high-
performance liquid chromatography (HPLC) (20-23). As these
chromatographic methods require a number of clean-up proce-
dures prior to determination, they are laborious and time-
consuming, and they require sophisticated equipment available
only in well-equipped centralized laboratories. Furthermore,
using a large amount of organic solvents raises concern
regarding health hazards to analysts and creation of environ-
mental pollution, and there are limitations on the capacity of
throughput of samples.
MATERIALS AND METHODS
Chemicals and Instrumentation. Triclopyr and the structurally
related pesticides used in cross-reaction studies were of analytical grade
and were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan),
Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Dr. Ehrenstorfer
(Augsburg, Germany), and Riedel-de Hae¨n (Seelze, Germany). 3,5,6-
Trichloro-2-pyridinol was produced from chlorpyrifos as mentioned
in the Hapten Synthesis section below. All organic starting materials
for hapten synthesis were purchased from Wako Pure Chemical
Industries, Ltd., and were of reagent quality or better. 6-(2,4-
Dinitrophenyl)aminohexanoic acid (Nꢀ-2,4-DNP-L-lysine hydrochloride)
was from Sigma Chemical Co. (St. Louis, MO). Thin-layer chroma-
tography (TLC) was performed using 0.2-mm precoated silica gel 60
F254 on glass plates from Merck (Darmstadt, Germany), and Rf values
refer to TLC with visualization under exposure to either ultraviolet
light or iodine vapor stain. Flash chromatographic separations were
carried out on C18 (38-63 µm particle size) (Wako). Bovine serum
albumin (BSA, fraction V), chicken egg ovalbumin (OVA), goat anti-
rabbit immunoglobulin (IgG) conjugated to horseradish peroxidase
(HRP), o-phenylenediamine (OPD) tablets, and complete and incom-
plete Freund’s adjuvants were from Sigma Chemical Co. Block Ace
was from Dainippon Chemical Industries (Osaka, Japan). Water used
in ELISA tests was purified using a Milli-Q system (Millipore Corp.,
Milford, MA). The ELISAs were carried out in 96-well polystyrene
microplates (Sumitomo Bakelite Co., Ltd., Tokyo, Japan). Proton
nuclear magnetic resonance (1H NMR) spectrum was obtained in
deuteriochloroform (CDCl3) or deuteriodimethyl sulfoxide (DMSO-
d6) with tetramethylsilane (TMS) as an internal standard on a JEOL
GSX270F instrument at 270 MHz and are described as multiplicity,
coupling constant (J) in hertz (Hz), number of protons, and assignment.
Chemical shift values (δ, ppm) are reported downfield from TMS.
ELISAs were analyzed using a Bio-Rad model 550 microplate reader
(Hercules, CA).
Hapten Synthesis. Synthesis of the hapten was carried out as
outlined in Figure 2. A new concept of hapten, that is, bifunctional
hapten, was synthesized by coupling between monofunctional hapten,
triclopyr, and lysine with a 2,4-dinitrophenyl (DNP) group.
Methyl 2-Amino-6-(2,4-dinitrophenyl)aminohexanoate (A, Figure 2).
In a 100-mL three-neck flask, 2 g of Nꢀ-2,4-DNP-L-lysine hydrochloride
(6.4 mmol) was dissolved in 40 mL of methanol, and the mixture was
stirred in an ice bath. Furthermore, 4 mL of thionyl chloride (56.2
mmol) was added, and the reaction mixture was stirred overnight at
room temperature. At the end of the reaction, the solvent was removed
under reduced pressure, and the residual crystals were recrystallized
from ether to give 2.06 g, a 99.1% yield of yellow crystalline. 1H NMR
(DMSO-d6) δ 1.36-1.93 (m, 7H, NHCH2CH2CH2), 3.15-3.29 (m,
2H, CH2CH), 3.51 (bs, 2H, NH2), 3.75 (s, 3H, OCH3), 4.01-4.17 (m,
1H, CHCO), 8.76-8.89 (m, 3H, Ar).
In the 1980s Hammock and Mumma (24) advocated the
adaptability of immunoassays (enzyme-linked immunosorbent
assays, ELISAs) for monitoring pesticides in various matrixes
such as environmental media, foods, and so on. Immunoassays
are simple, fast, cost-effective, and adaptable to on-site, high-
sample-throughput analyses (25, 26). They have been developed
both as screening tools and as quantitative analytical methods

New Bifunctional Hapten for Herbicide Immunoassay
J. Agric. Food Chem., Vol. 50, No. 13, 2002 3639
Table 1. Selected Indirect Competitive ELISA Screening Data against
Triclopyr and 3,5,6-Trichloro-2-pyridinola
antiserum
coating antigen
IC50 (nM)
triclopyr immunogen
LysTCPY-BSA
TCPY-BSA
R-20
LysTCPY-OVA
TCPY-OVA
245T-OVA
TCPY-OVA
245T-OVA
3.5
4.2
6.8
15.8
20.9
R-21
3,5,6-trichloro-2-pyridinol immunogen
LysTCPY-BSA
TCPY-BSA
R-20
LysTCPY-OVA
TCPY-OVA
256T-OVA
TCPY-OVA
245T-OVA
5.1
9.8
14.5
18.6
24.9
R-21
a Each standard was prepared in a 5% methanol/PBS solution.
hydroxide. The mixture was neutralized with 2 M HCl, and the product
was extracted using chloroform (2 × 30 mL). After the chloroform
phase was dried over anhydrous sodium sulfate, chloroform was
removed under reduced pressure. The residual crystals were recrystal-
lized from n-hexane to give 384.6 mg, a 91.7% yield of white needles.
Hapten Conjugation. The synthesized bifunctional hapten (LysTCPY,
Figure 1), and the commercial triclopyr (TCPY, Figure 1), and 2,4,5-T
(245T, Table 2) with a carboxylic acid functional group were coupled
covalently to proteins by active ester method according to the procedure
described by Karu et al. (35). Each hapten (0.1 mmol) was dissolved
in 500 µL of dry DMF, and then 11.5 mg of N-hydroxysuccinimide
(NHS) (0.1 mmol) and 20.6 mg of DCC (0.1 mmol) were added. The
reaction mixture was stirred for 4 h at room temperature. The precipitate
was removed by centrifugation. A 25-mg portion of BSA or OVA was
dissolved in 2.5 mL of 10 mM phosphate-buffered saline (PBS; 1.1
g/L Na2HPO4, 0.306 g/L KH2PO4, 0.9% (w/v) NaCl, pH 7.2) and 525
µL of dry DMF. Aliquots (125 µL each) of the activated hapten solution
were added dropwise to the two stirred protein solutions. The reaction
mixture was stirred overnight at 4 °C, and then dialyzed against PBS
(4×, 3 L each) overnight at 4 °C. The purified conjugates were
lyophilized, and stored at 4 °C.
Polyclonal Antiserum Production. System 1. For production of
group-specific polyclonal antiserum to triclopyr and 3,5,6-trichloro-2-
pyridinol, a female Japan white rabbit (about 3 kg, Tokyo Animal
Laboratory Inc., Tokyo, Japan) was immunized by intradermal injection
in footpad sites with 1.0 mg of LysTCPY-BSA dissolved in 0.5 mL
of PBS and emulsified with 0.5 mL of complete Freund’s adjuvant.
For the booster immunization, 0.5 mg of LysTCPY-BSA in 0.5 mL
of PBS/0.5 mL of incomplete Freund’s adjuvant was used. The booster
immunization was made intradermally and subcutaneously at multiple
sites on the back of the rabbit, and was performed at 2-week intervals
after the initial immunization. The booster immunization was performed
three times. The rabbit was bled from the marginal ear vein, and the
hapten-specific titers of the obtained antisera were monitored. A week
after the last injection, the rabbit was bled, and the antisera were
collected and stored at -80 °C.
System 2. A female Japan white rabbit was immunized with 1.0 mg
of TCPY-BSA. This immunization method was similar to that
described above for system 1. The booster immunization (0.5 mg of
TCPY-BSA) was repeated three times at 2-week intervals and then
monthly.
Determination of Antiserum Titer. Triclopyr and 3,5,6-trichloro-
2-pyridinol-specific polyclonal antiserum titer was determined by
checkerboard titration based on indirect ELISA. Dilutions of coating
antigens and antisera were chosen to produce an absorbance at 490
nm of approximately 0.5 after 30 min incubation at room temperature.
Indirect Competitive ELISA. Microplates were coated overnight
at 4 °C with 100 µL of 0.1 M carbonate-bicarbonate buffer (pH 9.6)
containing 62.5 ng of coating antigen. After the plates had been washed
with washing solution (PBS) by using a Nunc-Immuno Wash 8
microplate washer (Nalge Nunc International, Roskilde, Denmark), the
surface of the wells was blocked with 300 µL/well of blocking solution
Figure 2. Synthetic pathway of bifunctional hapten, LysTCPY. HOBt,
1-hydroxy-1H-benzotriazole monohydrate; DCC, N,N′-dicyclohexylcarbo-
diimide; TEA, triethylamine.
Methyl 2-[[[(3,5,6-Trichloro-2-pyridyl)oxy]methylcarbonyl]amino]-
6-(2,4-dinitrophenyl)aminohexanoate (B, Figure 2). In a 100-mL three-
neck flask, 500 mg of triclopyr (2.37 mmol), 0.73 g of 1-hydroxy-1H-
benzotriazole monohydrate (HOBt), and 0.86 g of A (2.37 mmol) were
dissolved in 10 mL of THF, and then 330 µL of triethylamine (2.37
mmol) and 0.51 mg of N,N′-dicyclohexylcarbodiimide (DCC) (2.49
mmol) dissolved in 772 µL of THF were added to the mixture. The
reaction mixture was stirred for 1 h in an ice bath and for 3 h at room
temperature. At the end of the reaction, 50 mL of ethyl acetate was
added, and the resultant mixture was filtered to remove dicyclohexyl-
urea. After ethyl acetate was removed under reduced pressure, the
resultant residue was reconstituted with 50 mL of ethyl acetate, and
then the ethyl acetate phase was washed with saturated sodium
bicarbonate solution, saturated ammonium chloride solution, saturated
sodium bicarbonate solution, and water (50 mL of each). After the ethyl
acetate phase was dried over anhydrous sodium sulfate, ethyl acetate
was removed under reduced pressure. The resultant residue was purified
with flash chromatography on C18 (6 g) (methanol/water, 1:1 v/v). After
the eluted fraction was collected and lyophilized, the yellow crystalline
was obtained in 57.1% yield (702 mg). 1H NMR (DMSO-d6) δ 1.03-
1.83 (m, 7H, NHCH2CH2CH2), 3.63 (s, 3H, OCH3), 4.30-4.39 (m,
1H, CHCO), 4.78 (s, 2H, COCH2), 4.82-4.94 (q, J ) 15.0 Hz, 2H,
CH2CH), 8.39 (s, 1H, pyridine-H), 8.55-8.88 (m, 3H, Ar).
2-[[[(3,5,6-Trichloro-2-pyridyl)oxy]methylcarbonyl]amino]-6-(2,4-
dinitrophenyl)aminohexanoic acid (LysTCPY, Figures 1 and 2). The
hydrolysis reaction of B (100 mg, 0.2 mmol) was conducted in 3 mL
of 1,4-dioxane/water (1:2 v/v) with 5 equiv of lithium hydroxide
monohydrate. After 2 h, the resultant reaction mixture was acidified to
pH 4 with 2 M HCl. After the resultant mixture was lyophilized, the
1
yellow crystalline was obtained in 90.9% yield (88.4 mg). H NMR
(DMSO-d6) δ 1.03-1.96 (m, 7H, NHCH2CH2CH2), 3.92-4.09 (m,
1H, CHCO), 4.33 (s, 2H, COCH2), 4.73-4.86 (q, J ) 14.8 Hz, 2H,
CH2CH), 8.37 (s, 1H, pyridine-H), 8.59-8.98 (m, 3H, Ar).
3,5,6-Trichloro-2-pyridinol. This compound was produced as de-
scribed by Beasely et al. (34) with slight modifications. Chlorpyrifos
(742.5 mg, 2.13 mmol) was hydrolyzed by refluxing for 4 h in 9 mL
of 70% (v/v) ethanol in water containing 394 mg of potassium

3640 J. Agric. Food Chem., Vol. 50, No. 13, 2002
Watanabe et al.
Table 2. Cross-Reactivity (CR) to Triclopyr and Related Compounds with Antiserum 20a
a Concentrations for assay were as follows: immobilized concentration of coating antigen, LysTCPY-OVA (62.5 ng/well); antiserum R-20 (1:6000, final dilution in wells).
Preparation of assay conditions was as described in the Materials and Method section. b CR ) (IC of triclopyr/IC of related compound) × 100. IC is the analyte
50
50
50
concentration that reduces the assay signal to 50% of the maximum value.
(25% (v/v) Block Ace in distilled water containing 0.1% (w/v) sodium
azide) by incubation for 2 h at room temperature to minimize
nonspecific binding in the plate. The plates were washed and incubated
with antiserum diluted with PBS for 1 h at room temperature. For this
step of the competitive ELISA, the plates were incubated with a mixture
of a constant concentration of antiserum with various concentrations
of analytes. The plates were washed and further incubated with goat
anti-rabbit IgG-HRP (1:8000 in PBS, 100 µL/well) for 1 h at room
temperature. The plates were washed again, and 100 µL of substrate
solution (2.0 mg/mL OPD and 0.02% (v/v) H2O2 in phosphate-citrate
buffer, pH 5.2) was added to each well. After 30 min, the reaction was
stopped with 0.5 M sulfuric acid, and the absorbance at 490 nm was
read and recorded. The intensity of color was inversely proportional
to the concentration of free triclopyr or 3,5,6-trichloro-2-pyridinol.
Effects of Organic Solvents. The effects of organic solvents were
tested by dissolving the analyte in PBS containing various proportions
of solvent (1, 5, 10, 20, and 30% (v/v) final concentration of each
organic solvent) and incubating these with antiserum in PBS on the
coating plate. Methanol, acetone, and acetonitrile were tested in this
study.
Cross-Reactivity (CR). The ability of the obtained antiserum to
recognize several structurally related compounds was tested by

New Bifunctional Hapten for Herbicide Immunoassay
J. Agric. Food Chem., Vol. 50, No. 13, 2002 3641
Figure 3. Antisera titers obtained with bifunctional hapten, LysTCPY-BSA and conventional hapten, TCPY-BSA. The antiserum R-20 titer obtained from
rabbit immunized with LysTCPY-BSA is shown by solid symbols, and the antiserum R-21 titer obtained from rabbit immunized with TCPY-BSA is shown
by open symbols. The antisera titers were estimated by indirect ELISA with each antiserum diluted at 1:6000 (final dilution in wells) and each coating
antigen immobilized at 125 ng/well. The days (x axis) shown are the total number of days after first immunization. The booster injection was performed
3 or 5 times after first immunization.
performing competitive assays and determining their respective IC50
(nanomolar) values (analyte concentration that reduces the maximum
signal of the competitive ELISA to 50%). CR value was calculated as
[IC50(triclopyr)/IC50(related compound)] × 100.
interesting function of the DNP group, and applied it to antibody
production for low molecular compound. The final goal in this
study was to obtain antiserum in a shorter period by using
bifunctional hapten. The previously described LysMNPA for
fenitrothion and its metabolite consisted of lysine derivatized
at its R-amino group with 3-methyl-4-nitrophenoxyacetic acid,
and derivatized at its ꢀ-amino group with DNP group. The
LysTCPY described here has an analogue structure. The amide
and carbonyl groups are thought to make the spacer arm stiffer
and more planar, allowing more space for the lysine R-carboxyl
group to be covalently coupled to the carrier protein.
Water Sample Analysis. The optimized ELISA was applied to
triclopyr or 3,5,6-trichloro-2-pyridinol determinations in different water
samples. Water samples were fortified with triclopyr or 3,5,6-trichloro-
2-pyridinol to evaluate potential matrix effects in ELISAs. The waters
tested were Milli-Q-purified water, tap water, a commercial bottled
water, and samples from Tama River (Tokyo, Japan). The river water
was collected in 1-L bottles and stored at 4 °C until required. For ELISA
analysis, 10 mL of water was spiked with known concentrations of
each targeted compound covering the quantitative working range. Tap
water and river water samples were filtered through a 0.45-µm nylon
filter and adjusted to pH 7.2 with PBS. Aliquots (500 µL) were then
mixed with 500 µL of antiserum (1:6000 in PBS), and used in the
ELISA. Analyte concentrations were interpolated from response curves
for reference standards in PBS.
Comparison of Immunoresponse to Conjugate of each
Hapten. To evaluate the promotive effect of the DNP group
introduced into LysTCPY in the immunized animal, the titer
of the antiserum R-20 obtained from the rabbit immunized with
LysTCPY-BSA (System 1) was compared with the titer of the
antiserum R-21 from the rabbit immunized with the monofunc-
tional hapten, TCPY-BSA (System 2). The titer of the antiserum
R-20 was regularly monitored against three kinds of coating
antigens, and the titer of the antiserum R-21 was regularly
monitored against two kinds of coating antigens using check-
erboard titration. As shown in Figure 3, the titer of the antiserum
R-20 against the homologous hapten, LysTCPY-OVA, was
remarkably increased after the first booster immunization, and
the one against heterologous hapten, TCPY-OVA, was linearly
increased after the second booster immunization. On the other
hand, the increase of the titer of the antiserum R-21 was clearly
later than the one of the antiserum R-20, and it took 147 days
from the primary immunization to come to same titer level as
that of the antiserum R-20 after 63 days. Although this result
is the increase of the titer against homologous hapten, TCPY-
OVA, the one against heterologous hapten, 245T-OVA, was
still insufficient for triclopyr or 3,5,6-trichloro-2-pyridinol
RESULTS AND DISCUSSIONS
Hapten Synthesis. Triclopyr and 3,5,6-trichloro-2-pyridinol
have a common structure, that is, a 3,5,6-trichloro-2-pyridyloxy
group in their molecules (Figure 1). In the present study, to
obtain group-specific antiserum to triclopyr and 3,5,6-trichloro-
2-pyridinol, we paid attention to this common structure, and a
bifunctional hapten with a DNP group was designed and
synthesized as shown in Figures 1 and 2. This hapten evoked
an antiserum which group-specifically reacted to fenitrothion
and its related compounds, fenitrooxon and 3-methyl-4-nitro-
phenol, only 40 days after the primary immunization. The
function of the DNP group is the promotive effect of ability of
antibody production in animals as reported by Eisen and Siskind
(36). On the other hand, Goodman et al. (37) reported a unique
bifunctional antigen introduced DNP group to activate T-
lymphocyte responses. In the present study, we considered the

3642 J. Agric. Food Chem., Vol. 50, No. 13, 2002
Watanabe et al.
measurement. From these findings, the DNP group introduced
into LysTCPY had the promotive effect to the ability of antibody
production in rabbit as might have been expected. It was
successful in obtaining the antiserum with enough titer for
fenitrothion or its related compounds measurement only 40 days
after the primary immunization when the group-specific anti-
serum to these compounds was produced based on the concept
in our previous report [unpublished data]. Thus, in the present
results as well as the previous development of antiserum to
fenitrothion and its metabolites, the bifunctional haptens with
a DNP moiety made it possible to develop a strong antibody
response in a shorter time, with fewer boosts.
Screening and Selection of Combination of Immuno-
reagents. In this section, the antiserum R-20 obtained from
rabbit immunized with LysTCPY-BSA after 63 days from the
primary immunization, and the antiserum R-21 obtained from
rabbit immunized with TCPY-BSA after 147 days from the
primary immunization were used to screen against each coating
antigen. Furthermore, combinations of coating antigen and an
antiserum having an optical density of >0.5 were selected, and
then subjected to competitive inhibition experiments against
triclopyr and 3,5,6-trichloro-2-pyridinol. When both the antisera
R-20 and R-21 were diluted at 1:6000 (final dilution in wells),
and the immobilized concentration of three kinds of coating
antigens was 62.5 ng/well, the optimal absorbances were shown.
On the basis of the conditions shown in Table 1 competitive
inhibition to triclopyr and 3,5,6-trichloro-2-pyridinol was per-
formed. The homologous assay using LysTCPY-OVA as the
coating antigen had a higher sensitivity to both compounds than
the homologous assay using TCPY-OVA as the coating antigen.
Furthermore, in the case of the heterologous assay using 245T-
OVA changed from pyridyl nitrogen to carbon, the sensitivity
to triclopyr and 3,5,6-trichloro-2-pyridinol was roughly 2-3
times more sensitive (lower IC50 value) than that of the
homologous assay. The optimum ELISA was obtained using
antiserum R-20 diluted 1:6000 and LysTCPY-OVA (62.5 ng/
well) as the coating conjugate.
Figure 4. ELISA inhibition curve for triclopyr (O) and 3,5,6-trichloro-2-
pyridinol (0). The data are corrected for background and are averages
of three replicates with coefficient of variation below 13%. Concentrations
for assay were as follows: immobilized concentration of coating antigen,
LysTCPY-OVA, 62.5 ng/well; antiserum R-20, 1:6000, final dilution in wells;
assay buffer, 10 mM PBS, pH 7.2; incubation time and temperature of
each competition step, 1 h at room temperature.
were sensitive enough for practical detection of triclopyr, 2,4,5-
T, and 3,5,6-trichloro-2-pyridinol.
Potential ELISA Interferences. Various matrixes may
interfere with the antigen-antibody interaction, causing differ-
ences between expected and observed ELISA results, and poor
correlation with instrumental analysis. Accordingly, we studied
analyte solubility, pH, concentration of organic solvents, and
the presence of Tween 20 surfactant as possible sources of
interference in ELISAs with antiserum R-20.
Organic SolVent Effects. Methanol (15, 20-22), acetone, and
acetonitrile (20, 22), which are commonly used to extract
analytes from various matrixes or to elute analytes from solid-
phase extraction (SPE) cartridges, were tested for their effects
on the ELISA. The effects on hapten binding and assay
sensitivity were measured as changes in the binding endpoint
(maximum absorbance at 490 nm; Amax), and changes in IC50
value of a competition ELISA were both reduced (IC50
increased) when organic solvent concentration was increased
(Figure 5). Methanol had the least effect and could be included
at concentrations up to 10% (v/v).
Effect of pH. Many immunoassays are equally sensitive over
a wide range of pH values. However, the carboxylic group on
triclopyr and the hydroxyl group on 3,5,6-trichloro-2-pyridinol
are potentially susceptible to ionization resulting from pH
changes. To examine the influence of pH on the ELISA,
competitive binding curves for each compound were obtained
at pHs from 5.2 to 10.2. As shown in Figure 6, on the triclopyr
ELISA, Amax did not change significantly in the tested pH
change. On the other hand, IC50 value was remarkably increased
at basic pH, that is, the sensitivity of the antiserum R-20 was
about two times lower than at neutral or acidic pH. As shown
in Figure 6, the 3,5,6-trichloro-2-pyridinol ELISA was very
sensitive to pH, that is, Amax and IC50 values significantly
changed at acidic and basic pH, and the sensitivity of the
antiserum R-20 was about two times lower than at pH 7.2. On
the basis of these results, pH 7.2 showing the highest affinity
of the antiserum R-20 and the maximum absorbance, was
selected as the optimum pH at which to carry out the ELISA
for triclopyr and 3,5,6-trichloro-2-pyridinol.
Cross-Reactivity (CR). The specificity of antiserum R-20
was evaluated with compounds of closely related molecular
structure (such as phenoxyacetic acid derivatives, organophos-
phorus insecticides, and phenolic compounds): their respective
IC50 values were obtained, and these data were compared with
triclopyr IC50 value. CR values for each compound are given
in Table 2. Antiserum R-20 showed 75% CR with 2,4,5-T,
which has a carbon instead of the pyridyl nitrogen. Silvex, which
also has the 2,4,5-trichlorination pattern, was 33% cross-reactive,
indicating that the methyl group at the R-carbon caused
relatively little hindrance of binding. By contrast, 2,4-D was
only 3% cross-reactive, indicating that chlorine at the 5-position
was very important for evoking the desired antibody. Dichlor-
prop, which lacks the 5-chlorine and has the methyl group at
the R-carbon, had virtually no cross-reaction with antiserum
R-20. Mecoprop, which has methyl groups on 2-position of the
aromatic ring and on the R-carbon, and MCPA and MCPB,
which are methylated at 2-position, all lack chlorination at
5-position, and their CR with antiserum R-20 is negligible.
Chlorpyrifos and chlorpyrifos-methyl, with a 3,5,6-tichloro-2-
pyridiyloxy group, had less than 9% CR, and no significant CR
was found for the organophosphorus insecticides (dichlo-
fenthion, bromophos-ethyl, and bromophos). The CR was about
69% for 3,5,6-trichloro-2-pyridinol. Together, these results are
consistent with the hypothesis that LysTCPY evoked both types
of antibodies in antiserum R-20. ELISAs with antiserum R-20

New Bifunctional Hapten for Herbicide Immunoassay
J. Agric. Food Chem., Vol. 50, No. 13, 2002 3643
Figure 5. Influence of different organic solvents on the analytical parameters of the triclopyr (open symbols) and 3,5,6-trichloro-2-pyridinol (solid symbols)
indirect competitive ELISAs. Data were obtained from standard curves performed in two replicates in buffers of different concentrations of each organic
solvent (O or b, methanol; 0 or 9, acetone; ] or [, acetonitrile). Results are the mean of three independent experiments.
Figure 6. Influence of the assay buffer pH on the analytical characteristics of triclopyr (O) and 3,5,6-trichloro-2-pyridinol (0) competitive standard curves.
The assay conditions were as follows: immobilized coating antigen, LysTCPY-OVA, 62.5 ng/well; antiserum R-20, 1:6000, final dilution in wells; incubation
time and temperature of each competition step, 1 h at room temperature. The data are corrected for the background and are the average of two
replicates.
ImproVement of Matrix Effect and Effect of Tween 20. The
influence of the matrix effects originated from four kinds of
water samples was evaluated. As mentioned above, the maxi-
mum merits of ELISA are that it is possible to substantially
reduce the labor of sample preparation such as concentration,
SPE, or liquid-liquid partition only extraction procedure from
samples. In the present study, each water sample was directly
analyzed by very simple sample preparation in which the sample
was diluted only with an equal volume of the antiserum R-20
solution. As shown in Figures 7 and 8, on both triclopyr and
3,5,6-trichloro-2-pyridinol ELISAs, a matrix effect was observed
in river and tap water samples, that is, the competitive curves
obtained from river water and tap water samples shifted to higher
absorbance than the PBS control curve. To correct the matrix
effect, it is well-known that the usage of an additive such as
the nonionic detergent Tween 20 (32, 38-40) or BSA (41) is
very effective in suppressing nonspecific adsorption, so Tween
20 was selected as an additive. Tween 20 was investigated
concerning the improvement of matrix effect and the influence
on the sensitivity of the antiserum R-20. As shown in Figures
7 and 8, the matrix effects observed in river and tap water
samples were improved by addition of 0.05% (v/v) Tween 20,
and the addition of Tween 20 did not affect the sensitivity of
the antiserum R-20. These results suggest that it is possible to
analyze real water samples as the optimal condition in which
the 0.05% (v/v) Tween 20 was added to dilution buffer.
Analytical Parameters of the Optimized Triclopyr and
3,5,6-Trichloro-2-pyridinol ELISAs. The optimized triclopyr
and 3,5,6-trichloro-2-pyridinol ELISAs used coating antigen
LysTCPY-OVA at 62.5 ng/well, the antiserum R-20 at a dilution
of 1:6000 (final dilution in wells), and 0.05% (v/v) PBST as
dilution buffer (Table 3). The assay affinity for triclopyr and
3,5,6-trichloro-2-pyridinol, represented by the IC50 values, were
0.72 and 0.95 ng/mL, respectively (Table 3).
There is not a general agreement for the calculation of assay
sensitivity and working range of competitive immunoassays
(42). Fleeker (43) reported the LOD of an assay to be three
times the standard deviation of the A0, negative control, from

3644 J. Agric. Food Chem., Vol. 50, No. 13, 2002
Watanabe et al.
Figure 7. Comparison of triclopyr competitive curves obtained from standards prepared in PBS not containing Tween 20 (A) and containing 0.05% (v/v)
Tween 20 (PBST) (B): (O) control; ([) river water; (4) tap water; (0) purified water; and (2) bottled water. The assay conditions were as follows:
immobilized coating antigen, LysTCPY-OVA, 62.5 ng/well; antiserum R-20, 1:6000, final dilution in wells; assay buffer, PBS (A) or 0.05% (v/v) PBST (B);
incubation time and temperature of each competition step, 1 h at room temperature; pH, 7.2. The data are corrected for the background and are the
average of two replicates. (A) IC values were as follows: control, 0.84 ng/mL; river water, 0.84 ng/mL; tap water, 0.8 ng/mL; purified water, 0.7 ng/mL;
50
bottled water, 0.75 ng/mL. (B) IC values were as follows: control, 0.87 ng/mL; river water, 0.75 ng/mL; tap water, 0.89 ng/mL; purified water, 0.8
50
ng/mL; bottled water, 0.82 ng/mL.
Figure 8. Comparison of 3,5,6-trichloro-2-pyridinol competitive curves obtained from standards prepared in PBS not containing Tween 20 (A) and
containing 0.05% (v/v) Tween 20 (PNST) (B): (O) control; ([) river water; (4) tap water; (0) purified water; and (2) bottled water. The assay conditions
were as follows: immobilized coating antigen, LysTCPY-OVA, 62.5 ng/mL; antiserum R-20, 1:6000, final dilution in wells; assay buffer, PBS (A) or 0.05%
(v/v) PBST (B); incubation time and temperature of each competition step, 1 h at room temperature; pH, 7.2. The data are corrected for the background
and are the average of two replicates. (A) IC values were as follows: control, 0.78 ng/mL; river water, 1.2 ng/mL; tap water, 1.25 ng/mL; purified water,
50
0.82 ng/mL; and bottled water, 0.8 ng/mL. (B) IC values were as follows: control, 0.76 ng/mL; river water, 0.75 ng/mL; tap water, 0.76 ng/mL; purified
50
water, 0.71 ng/mL; and bottled water, 0.8 ng/mL.
its mean absorbance, whereas Midgley et al. (44) calculated
the LOD as the concentration that corresponds to 90% of the
A/A0. For the optimized assay, the LOD was calculated
according to the method reported by Midgley et al., and the
quantitative working range was established between the con-
centrations producing 80% and 20% of the A/A0. Using these
criterions, the quantitative working range of the triclopyr ELISA
was from 0.1 to 5.2 ng/mL with a LOD of 0.037 ng/mL. On
the other hand, the quantitative working range of the 3,5,6-
trichloro-2-pyridinol ELISA was from 0.13 to 6.0 ng/mL with
a LOD of 0.052 ng/mL.
Applications to Water Samples. Spiking water samples with
several amounts of triclopyr or 3,5,6-trichloro-2-pyridinol is a
common practice to perform a preliminary evaluation of
analytical assay reliability. Some water samples from different
sources were spiked at several concentrations of triclopyr or
3,5,6-trichloro-2-pyridinol covering the optimized working range
(1, 5, and 10 ng/mL). Direct analysis of each spiked sample
without dilution or SPE such as a C18 cartridge resulted in
accurate determinations of triclopyr and 3,5,6-trichloro-2-
pyridinol concentrations. Determinations were made in quadru-
plicate, and the mean absorbance was used to estimate triclopyr

New Bifunctional Hapten for Herbicide Immunoassay
J. Agric. Food Chem., Vol. 50, No. 13, 2002 3645
In particular, on the triclopyr ELISA, the IC50 value is about
two times lower than that of the ELISA reported by Johnson
and Hall (29). In the final optimum conditions, assay precision
and accuracy in the presence of matrix effects from real water
samples were investigated. The satisfactory recoveries suggested
that the proposed ELISA for two compounds can be used for
the preliminary screening method in water samples.
Table 3. Recoveries of Triclopyr and 3,5,6-Trichloro-2-pyridinol from
Spiked Water Samples Measured by the Optimized ELISAs
b
c
added
(ng/mL)
recovereda
(ng/mL)
SD
CV
recovery
(%)
sample
(ng/mL)
(%)
triclopyr
1.1
4.9
9.8
1.0
5.1
10.4
1.1
4.9
10.0
1.0
river water
1
5
10
1
5
10
1
5
10
1
5
10
0.141
0.250
0.472
0.103
0.311
0.436
0.126
0.340
0.479
0.086
0.377
0.275
12.9
5.1
4.8
10.2
6.2
4.2
11.7
6.9
4.8
8.8
7.3
2.7
7.1
110.0
97.5
97.8
101.3
101.0
103.5
107.5
98.5
99.8
97.8
103.5
100.8
101.6
ABBREVIATIONS USED
tap water
Amax, maximum absorbance in the absence of competing
analyte; BSA, bovine serum albumin; CR, cross-reactivity; CV,
coefficients of variation; DCC, N,N′-dicyclohexylcarbodiimide;
DMF, N,N-dimethylformamide; DMSO, N,N-dimethyl sulfox-
ide; DNP, 2,4-dinitrophenyl; ECD, electron capture detector;
ELISA, enzyme-linked immunosorbent assay; GC, gas chro-
matography; 1H NMR, proton nuclear magnetic resonance;
HOBt, 1-hydroxy-1H-benzotriazole monohydrate; HPLC, high-
performance liquid chromatography; HRP, horseradish peroxi-
dase; IC50, concentration giving 50% inhibition of maximum
response; IgG, immunoglobulin; LOD, limit of detection;
LysMNPA, 2-[[[(3-methyl-4-nitrophenyl)oxy]methylcarbonyl]-
amino]-6-(2,4-dinitrophenyl)aminohexanoic acid; LysTCPY,
2-[[[(3,5,6-trichloro-2-pyridinyl)oxy]methylcarbonyl]amino]-6-
(2,4-dinitrophenyl)aminohexanoic acid; MCPA, 4-chloro-o-
tolyloxyacetic acid; MCPB, 4-(4-chloro-o-tolyloxy)butyric acid;
NHS, N-hydroxysuccinimide; OPD, o-phenylenediamine; OVA,
ovalbumin; PBS, 10 mM phosphate buffer, 0.9% (w/v) NaCl,
pH 7.2; PBST, PBS containing 0.05% (v/v) Tween 20; SD,
standard deviation; SPE, solid-phase extraction; 245T, 2,4,5-
T; TCPY, triclopyr; TEA, triethylamine; THF, tetrahydrofuran;
TLC, thin-layer chromatography; TMS, tetramethylsilane.
purified water
bottled water
5.2
10.1
mean
3,5,6-trichloro-2-pyridinol
river water
1
5
10
1
5
10
1
5
10
1
5
10
1.0
4.9
9.7
1.0
5.0
10.0
1.0
4.8
10.1
0.9
5.3
0.183
18.3
10.1
7.5
12.3
7.0
8.0
11.5
7.0
8.2
13.9
6.7
3.2
9.5
100.0
98.0
97.0
104.5
99.5
100.0
100.0
95.0
100.8
92.0
106.0
104.3
99.8
0.497
0.730
0.128
0.350
0.804
0.115
0.332
0.822
0.128
0.356
0.330
tap water
purified water
bottled water
mean
10.4
a Each determination was run in quadruplicate, and the mean absorbance was
interpolated from a standard curve performed in the same ELISA plate. Data are
the average of 3 independent determinations. b Standard deviation. c Intra-assay
coefficient of variation.
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Received for review November 5, 2001. Revised manuscript received
April 2, 2002. Accepted April 11, 2002.
JF0114684