Development of an immunoassay for the residues of the herbicide bensulfuron-methyl.

Article abstract of DOI:10.1021/jf011150b

To develop a competitive indirect enzyme-linked immunosorbent assay based on polyclonal antibodies for the detection of the sulfonylurea herbicide bensulfuron-methyl, seven structurally related haptens were synthesized. Four of them mimicking the target analyte were conjugated to keyhole limpet hemocyanin by the N-hydroxysuccinimide activated ester method to use as immunogens, and all of them were conjugated to bovine serum albumin to use as plate-coating antigens. Polyclonal antibodies raised in rabbits and the coating antigens were screened and selected for the assay in simple homologous and heterologous ELISA formats. Three sensitive heterologous ELISAs were selected and optimized, showing the average IC(50) values of bensulfuron-methyl as low as 0.17, 0.09, and 0.09 ng/mL, the detection ranges of 0.04-0.60, 0.01-0.60, and 0.04-0.25 ng/mL, and the lowest detection limits of 0.03, 0.002, and 0.03 ng/mL, respectively. The cross-reactivities of other sulfonylurea herbicides and metabolites of bensulfuron-methyl to the antibodies were less than 15% in the two assays. Recoveries from the analyte-fortified water samples in assay I were in the range of 81-125% by simple dilution. The correlation between the ELISA and HPLC was 0.999 (n = 15) with a slope of 1.37 in the analysis of groundwater samples fortified with bensulfuron-methyl. The results obtained strongly indicate that the ELISA can be a highly sensitive and convenient tool for detecting bensulfuron-methyl residues in agricultural and environmental samples.

Full text of DOI:10.1021/jf011150b

J. Agric. Food Chem. 2002, 50, 1791−1803 1791
Development of an Immunoassay for the Residues of the
Herbicide Bensulfuron-Methyl
JAE KOO LEE,*,† KI CHANG AHN,† OEE SOOK PARK,‡ YONG KWAN KO,§ AND
§
DAE-WHANG KIM
Departments of Agricultural Chemistry and Chemistry, Chungbuk National University,
Cheongju 361-763, Korea, and Bio-Organic Division, Korea Research Institute of Chemical
Technology, Taejon 305-600, Korea
To develop a competitive indirect enzyme-linked immunosorbent assay based on polyclonal antibodies
for the detection of the sulfonylurea herbicide bensulfuron-methyl, seven structurally related haptens
were synthesized. Four of them mimicking the target analyte were conjugated to keyhole limpet
hemocyanin by the N-hydroxysuccinimide activated ester method to use as immunogens, and all of
them were conjugated to bovine serum albumin to use as plate-coating antigens. Polyclonal antibodies
raised in rabbits and the coating antigens were screened and selected for the assay in simple
homologous and heterologous ELISA formats. Three sensitive heterologous ELISAs were selected
and optimized, showing the average IC50 values of bensulfuron-methyl as low as 0.17, 0.09, and
0.09 ng/mL, the detection ranges of 0.04-0.60, 0.01-0.60, and 0.04-0.25 ng/mL, and the lowest
detection limits of 0.03, 0.002, and 0.03 ng/mL, respectively. The cross-reactivities of other sulfonylurea
herbicides and metabolites of bensulfuron-methyl to the antibodies were less than 15% in the two
assays. Recoveries from the analyte-fortified water samples in assay I were in the range of 81-
125% by simple dilution. The correlation between the ELISA and HPLC was 0.999 (n ) 15) with a
slope of 1.37 in the analysis of groundwater samples fortified with bensulfuron-methyl. The results
obtained strongly indicate that the ELISA can be a highly sensitive and convenient tool for detecting
bensulfuron-methyl residues in agricultural and environmental samples.
KEYWORDS: Bensulfuron-methyl; sulfonylurea herbicide; ELISA; polyclonal antibodies; monitoring
INTRODUCTION
17). Bioassays using some species, such as sugar beet, lentil,
or green alga, susceptible to sulfonylurea herbicides were also
reported (18-19).
Since their development in the 1970s, sulfonylurea herbicides
have been known to have many innovative properties. Because
of their low mammalian toxicity and very high herbicidal
activity, they have been in wide use to control broad-leaved
weeds and some grasses in a variety of crops (1). Their
extremely low application rate (as low as 2 g/ha), as well as
their chemical and thermal instability, requires a highly sensitive
analytical method for detecting their residues in environmental
and agricultural samples. The early analytical approaches utilized
normal-phase high-performance liquid chromatography (HPLC)
with photoconductivity detection (2). Because of their extremely
low volatility and thermal instability, they should be derivatized
(3-6), or hydrolyzed (7), prior to gas chromatography (GC).
The residues of some sulfonylurea herbicides in water (8-9)
and soil (10) were determined by capillary electrophoresis (CE).
Sensitive and selective immunoassays for some sulfonylurea
herbicides in soil and water were relatively recently described
for use with other complementary analytical techniques (11-
Bensulfuron-methyl, methyl 2-[[[[[(4,6-dimethoxypyrimidin-
2-yl)amino]carbonyl] amino]sulfonyl]methyl]benzoate, also be-
longs to the class of systemic sulfonylurea herbicides inhibiting
acetolactate synthase, a key enzyme in the biosynthesis of the
branched-chain amino acids of target plants. It is used for the
pre- and post-emergence control of annual and perennial broad-
leaved and sedge weeds in rice (1). The most common method
for the detection of bensulfuron-methyl involves HPLC with
either UV (20-21) or photoconductivity detection (22).
The instrumental methods such as HPLC, GC, and CE are
accurate, but they are expensive and time-consuming and need
much effort for sample preparation before analysis. Bioassay
is a sensitive tool for the sulfonylurea herbicides but may lack
selectivity, as it could encounter cross-reactivities by other
herbicidally active compounds that are chemically similar to
the target analyte. On the contrary, immunoassays generally are
rapid, sensitive, selective, and cost-effective. Recently, Kawada
et al. (15) reported on an ELISA for bensulfuron-methyl based
on monoclonal antibodies. Yuan et al. (17) also worked on a
fluoroimmunoassay for bensulfuron-methyl using a monoclonal
antibody.
* To whom correspondence should be addressed (telephone ++82-43-
261-2562; fax ++82-43-271-5921; e-mail jklee@cbucc.chungbuk.ac.kr).
† Department of Agricultural Chemistry.
‡ Department of Chemistry.
§ Korea Research Institute of Chemical Technology.
10.1021/jf011150b CCC: $22.00 © 2002 American Chemical Society
Published on Web 02/20/2002

1792 J. Agric. Food Chem., Vol. 50, No. 7, 2002
Lee et al.
TLC (ethyl acetate) Rf, 0.4 (tailing). 1H NMR (CDCl3): δ 7.95 (d, 1H,
J ) 7.5 Hz, Ar), 7.54-7.37 (m, 3H, Ar), 5.03 (s, 2H, CH2Cl), 4.34
(m, 2H, OCH2CH2CH2COOH), 2.55 (m, 2H, OCH2CH2CH2COOH),
2.49 (m, 2H, OCH2CH2CH2COOH).
The purpose of this investigation is to develop a competitive
indirect enzyme-linked immunosorbent assay (ciELISA) for
monitoring and efficiently quantifying bensulfuron-methyl
residues in agricultural and environmental samples, using
polyclonal antibodies produced against various immunogens
mimicking the analyte.
To a solution of the compound 4 (383 mg, 1.49 mmol) in 5 mL of
methanol was added thiourea (233 mg, 1.64 mmol). The mixture was
refluxed for 18 h, and concentrated on a rotary evaporator under reduced
pressure. The residue was purified by preparative TLC using ethyl
acetate as a developing solvent to give 383 mg of compound 5 as a
white solid. A suspension of the compound 5 (383 mg, 1.49 mmol) in
14 mL of methylene chloride/water mixture (1:1, v/v) was bubbled
with excess chlorine gas for 1 h at 0 °C. The resulting yellow mixture
was diluted with 30 mL of methylene chloride and then washed with
water (2 × 30 mL). The organic layer was dried over anhydrous
magnesium sulfate. The solvent was removed on a rotary evaporator
under reduced pressure to give 512 mg of compound 6 as a white
solid: 1H NMR (CDCl3): δ 8.02 (d, 1H, J ) 7.5 Hz, Ar), 7.63-7.49
(m, 3H, Ar), 5.62 (s, 2H, CH2SO2), 4.39 (m, 2H, OCH2CH2CH2COOH),
2.52 (m, 2H, OCH2CH2CH2COOH), 2.10 (m, 2H, OCH2CH2CH2-
COOH).
MATERIALS AND METHODS
Chemicals. Bensulfuron-methyl of analytical and working grade was
a gift from Du Pont. Bovine serum albumin (BSA), keyhole limpet
hemocyanin (KLH), goat anti-rabbit IgG peroxidase conjugate as the
second antibody, and Freund’s complete and incomplete adjuvants were
all obtained from Sigma Chemical Co. (St. Louis, MO). All reagents
for hapten synthesis were of analytical reagent grade and were used as
supplied. For the cross-reactivity study, nicosulfuron was supplied by
Ishihara Sangyo, azimsulfuron and thifensulfuron-methyl were supplied
by Du Pont, ethoxysulfuron was provided by Aventis, cyclosulfamuron
was from BASF, cinosulfuron was provided by Novartis, imazosulfuron
was supplied by Takeda, and pyrazosulfuron-ethyl was provided by
Nissan.
Instrument. 1H nuclear magnetic resonance (NMR) spectra of
synthesized compounds were obtained on a 300 MHz NMR spectrom-
eter, DPX300 (Bruker, Germany) using tetramethylsilane as an internal
standard. Infrared (IR) spectra were obtained on an FT-IR spectrom-
eter, IFS-66/FRA106S (Bruker, Germany). Fast atom bombardment
mass spectra (FABMS) by using 3-nitrobenzyl alcohol as a matrix were
obtained on a JEOL four sector tandem mass spectrometer, JMS-HX/
HX110A (JEOL, Japan), in the Korea Basic Science Institute. Melting
points were determined on a Gallenhamp melting point apparatus. Rf
To a cooled solution of the compound 6 (512 mg, 1.60 mmol) in 20
mL of ethyl acetate on an ice bath was added an aqueous solution of
25% ammonium hydroxide (0.25 mL). The reaction mixture was stirred
for 20 min at 0 °C. Thereafter, an excess of ammonium hydroxide
solution (0.2 mL) was again added. The mixture was stirred for 2 h
and then washed with 20 mL of 5% hydrochloric acid. The organic
layer was dried over anhydrous magnesium sulfate. The solvent was
removed by evaporation to give 31 mg of compound 8 as a white
solid: 1H NMR (CDCl3): δ 7.97 (d, 1H, J ) 7.5 Hz, Ar), 7.57-7.50
(m, 3H, Ar), 4.92 (s, 2H, CH2SO2), 4.77 (br, 2H, NH2), 4.41 (m, 2H,
OCH2CH2CH2COOH), 2.55 (m, 2H, OCH2CH2CH2COOH), 2.12 (m,
2H, OCH2CH2CH2COOH).
values refer to thin-layer chromatography (TLC) on silica gel 60 F254
,
precoated plates (Merck, Germany) with visualization under exposure
to either ultraviolet light (254 nm) or iodine vapor. Ultraviolet-visible
(UV/Vis) spectra were obtained on a U-2000 spectrometer (Hitachi,
Japan). HPLC analysis was carried out by using a Hewlett-Packard
1100 series HPLC equipped with a diode array detector. ELISA was
performed on 96-well microtiter plates (Nunc-Immuno plate, MaxiSorp
surface, Roskilde, Denmark) and read spectrophotometrically with a
microplate reader, Bio-Rad Model 550 (Hercules, CA).
To a stirred solution of the compound 8 (31 mg, 0.11 mmol) in 3
mL of acetonitrile were added phenyl (4,6-dimethoxypyrimidin-2-yl)-
carbamate (32 mg, 0.11 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU, 34 mg, 0.22 mmol). The reaction mixture was stirred at
room temperature for 30 min, diluted with ethyl acetate (30 mL), and
washed with 30 mL of 5% HCl. After the organic layer was dried over
anhydrous magnesium sulfate, the solvent was removed by a rotary
evaporator under reduced pressure. The residue was recrystallized from
diethyl ether/n-hexane mixture (1:1, v/v) to give 27 mg of pure hapten-2
as a white solid: TLC (methanol/methylene chloride/acetic acid, 1:9:
Hapten Synthesis and Verification. Hapten-1, 2-{[({[(4,6-
dimethoxypyrimidin-2-yl)amino]carbonyl}amino)sulfonyl]methyl}-
benzoic acid, bensulfuron. Hydrolysis of bensulfuron-methyl was
conducted to synthesize hapten-1. That is, bensulfuron-methyl (1000
mg, 2.44 mmol) dissolved in 30 mL of 1 N NaOH solution was stirred
at 65 °C until TLC showed no bensulfuron-methyl and the Na form of
bensulfuron with a low Rf value, compound 1. After 90 min the reaction
mixture was cooled and poured into 50 mL of water. The aqueous
solution was washed with 50 mL of ethyl acetate to remove some
impurities, acidified to pH 2 with 6 N hydrochloric acid, and then
extracted with ethyl acetate (2 × 50 mL). The combined organic layer
was dried over anhydrous magnesium sulfate and evaporated to dryness
under reduced pressure to give 850 mg of hapten-1 (bensulfuron) as a
white solid: TLC (methanol/methylene chloride/acetic acid)1:9:0.1,
v/v/v) Rf, 0.56; mp 186 °C. 1H NMR (DMSO-d6): δ 7.81 (d, 1 H, J )
7.5 Hz, Ar), 7.48-7.35 (m, 3H, Ar), 5.92 (s, 1H, pyrimidine H), 5.32
(s, 2H, CH2SO2), 3.73 (s, 6H, OCH3). IR (νmax, KBr) 3580-3210, 1718,
1654, 1457, 1364, 1160. Low FAB (+)-MS, m/z 397 [M + H]+.
Hapten-2, 4-[(2-{[({[(4,6-dimethoxypyrimidin-2-yl)amino]carbonyl}-
amino)sulfonyl] methyl}benzoyl)oxy]butanoic acid. To a cool, stirred
solution of 2-(chloromethyl)benzoyl chloride, 3 (2400 mg, 12.70 mmol)
in 20 mL of methylene chloride on an ice bath was added sodium
4-hydroxybutanoate (2160 mg, 15.22 mmol), together with triethylamine
(1540 mg, 15.22 mmol). Thereafter, 4-(dimethylamino)pyridine (DMAP,
7 mg, 0.64 mmol) was added at room temperature. The mixture was
stirred for 2 h, acidified with 20 mL of 5% HCl, and extracted with
methylene chloride (2 × 30 mL). The combined organic layer was
dried over anhydrous magnesium sulfate, and concentrated on a rotary
evaporator under reduced pressure. The residue was purified by silica
gel flash chromatography using ethyl acetate/n-hexane mixture (1:1,
v/v) as an eluent and by preparative TLC using ethyl acetate as a
developing solvent to give 383 mg of compound 4 as a white solid:
1
0.1, v/v/v) Rf, 0.56. H NMR (CDCl3): δ 7.91 (m, 1H, J ) 7.5 Hz,
Ar), 7.9 (br s, 1H, NHPy), 7.52-7.49 (m, 3H, Ar), 5.72 (s, 1H,
pyrimidine H), 5.39 (s, 2H, CH2SO2), 4.40 (m, 2H, OCH2CH2CH2-
COOH), 3.78 (s, 6H, OCH3), 2.53 (m, 2H, OCH2CH2CH2COOH), 2.03
(m, 2H, OCH2CH2CH2COOH). Low FAB (+)-MS, m/z 483.8 [M +
H]+.
Hapten-3, 6-[(2-{[({[(4,6-dimethoxypyrimidin-2-yl)amino]carbonyl}-
amino)sulfonyl] methyl}benzoyl)oxy]hexanoic acid. For the synthesis
of an ester 10, the compound 3 (1000 mg, 5.29 mmol) was added to 5
mL of methanol. The solution was refluxed for 3 h, and the solvent
was removed on a rotary evaporator under reduced pressure to give
1000 mg of methyl 2-(chloromethyl)benzoate, 10, as an oily product:
TLC (methylene chloride) Rf, 0.77.
Thiourea (413 mg, 5.42 mmol) was added to a solution of the
compound 10 (1000 mg, 5.42 mmol) in 10 mL of methanol and the
mixture was refluxed for 18 h. The resulting solution was concentrated
under reduced pressure and the residue was recrystallized twice from
ethyl acetate to give 1450 mg of compound 11 as a malodorous solid:
TLC (ethyl acetate) Rf, 0.08.
The suspension of the compound 11 (1450 mg) in 30 mL of
methylene chloride/water mixture (1:1, v/v) was bubbled with an excess
of chlorine gas at 0 °C for 1 h. The resulting mixture was diluted with
30 mL of methylene chloride and then washed twice with 20 mL of
water. The organic layer was dried over anhydrous magnesium sulfate
and concentrated. The residue was recrystallized from ethyl acetate and
then purified by filtration through a short pad of silica gel using ethyl
acetate/n-hexane mixture (3:7, v/v) as an eluent to give 1390 mg of
compound 12 as a white solid: TLC (ethyl acetate) Rf, 0.93. 1H NMR

ELISA for Bensulfuron-Methyl
J. Agric. Food Chem., Vol. 50, No. 7, 2002 1793
(CDCl3): δ 7.81 (d, 1H, J ) 7.5 Hz, Ar), 7.53-7.37 (m, 3H, Ar), 5.64
CH2CH2COOH), 1.52 (m, 2H, OCH2CH2CH2CH2CH2COOH), 1.37 (m,
2H, OCH2CH2CH2CH2CH2COOH). Low FAB (+)-MS, m/z 511.14 [M
+ H]+.
(s, 2H, CH2SO2), 3.89 (s, 3H, COOCH3).
The compound 12 (695 mg, 2.80 mmol) was dissolved in 30 mL of
ethyl acetate, and the solution was cooled on an ice bath. Tert-
butylamine (409 mg, 5.60 mmol) was added slowly to the solution.
The resulting white suspension was stirred at 0 °C for 1 h, and the
stirring was continued at room temperature for 2 h. The mixture was
washed with 30 mL of 5% hydrochloric acid. The organic layer was
dried over anhydrous magnesium sulfate and evaporated under reduced
pressure to give 440 mg of compound 13 as a white solid: TLC (ethyl
acetate/n-hexane, 1:1, v/v) Rf, 0.73.
Hapten-4,4-({[2-(methoxycarbonyl)benzyl]sulfonyl}amino)-4-oxobu-
tanoic acid. To 1 mL of trifluoroacetic acid was added the compound
13 (300 mg, 1.05 mmol). The mixture was stirred at room temperature
for 3 h and then evaporated to dryness. To the residue was added 30
mL of distilled water, and the mixture was extracted with ethyl acetate
(2 × 30 mL). The combined organic layer was dried over anhydrous
magnesium sulfate and concentrated on a rotary evaporator under
reduced pressure. The residue was recrystallized from ethyl acetate to
give 255 mg of compound 18 as a white solid: TLC (methylene
chloride/methanol, 1:9, v/v) Rf, 0.8. 1H NMR (CDCl3): δ 8.01 (d, 1H,
J ) 7.5 Hz, Ar), 7.61-7.40 (m, 3H, Ar), 4.90 (s, 2H, CH2SO2), 4.56
(s, 2H, SO2NH2), 3.94 (s, 3H, OCH3).
The hydrolysis of compound 13 (440 mg, 1.62 mmol) was conducted
in 20 mL of tetrahydrofuran (THF)/1 N NaOH mixture (1:1, v/v) at 65
°C for 5 h. THF was removed on a rotary evaporator under reduced
pressure. The alkaline solution was acidified with 20 mL of 5%
hydrochloric acid, and then extracted with ethyl acetate (2 × 30 mL).
The combined organic layer was dried over anhydrous magnesium
sulfate and concentrated on a rotary evaporator to give 270 mg of
compound 14 as a white solid: TLC (ethyl acetate/n-hexane, 1:1, v/v)
Hapten-4 was synthesized according to a method introduced by
Schlaeppi et al. (12). To 5 mL of acetonitrile was added 200 mg (0.87
mmol) of methyl-(aminosulfonyl)-o-toluate, compound 18, together with
87 mg (0.87 mmol) of succinic anhydride. Then, 265.5 mg (1.74 mmol)
of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was added dropwise for
10 min under slightly cooled conditions (22 °C). After this was stirred
for 2 h, the resulting solution was poured into 50 mL of distilled water
and washed with 50 mL of ethyl acetate to remove some impurities.
The organic layer was discarded, and the aqueous phase was acidified
to pH 2 with hydrochloric acid, and then extracted with ethyl acetate
(2 × 50 mL). The combined organic layer was dried over anhydrous
magnesium sulfate, the solvent was removed by evaporation, and the
residue was recrystallized from ethyl acetate to give 100 mg of hapten-4
as a white solid: TLC (methanol/methylene chloride/acetic acid, 1:9:
1
Rf, 0.22 (tailing). H NMR (CDCl3): δ 13.1 (br s, 1H, COOH), 7.81
(d, 1H, J ) 7.5 Hz, Ar), 7.53-7.37 (m, 3H, Ar), 4.88 (s, 2H, CH2-
SO2), 1.23 (s, 9H, NHC(CH3)3).
To a stirred solution of the compound 14 (1000 mg, 3.7 mmol) in
20 mL of acetonitrile were added 6-bromohexanoic acid (1008 mg,
5.6 mmol) and DBU (1127 mg, 7.4 mmol). After the reaction mixture
was heated at 50 °C for 1 h, it was concentrated on a rotary evaporator,
acidified with 30 mL of 5% hydrochloric acid, and extracted with ethyl
acetate (2 × 30 mL). The combined organic layer was dried over
anhydrous magnesium sulfate, and the solvent was removed on a rotary
evaporator. The residue was purified by using preparative TLC on silica
gel twice with methylene chloride/isopropyl alcohol mixture (9.5:0.5,
v/v) as a developing solvent to give 180 mg of compound 15 as a
white solid: TLC (methylene chloride/isopropyl alcohol, 9.5:0.5, v/v)
Rf, 0.4. 1H NMR (CDCl3): δ 7.91 (d, 1H, J ) 7.5 Hz, Ar), 7.60-7.30
(m, 3H, Ar), 4.88 (s, 2H, CH2SO2), 4.30 (m, 2H, OCH2CH2CH2CH2-
CH2COOH), 2.61 (br s, 1H, CH2SO2NH), 2.36 (m, 2H, OCH2CH2-
CH2CH2CH2COOH), 1.78 (m, 2H, OCH2CH2CH2CH2CH2COOH), 1.67
(m, 2H, OCH2CH2CH2CH2CH2COOH), 1.48 (m, 2H, OCH2CH2CH2-
CH2CH2COOH), 1.25 (s, 9H, NHC(CH3)3).
1
0.1, v/v/v) Rf, 0.47. H NMR (CD3OD): δ 7.91 (d, 1H, J ) 7.5 Hz,
Ar), 7.52-7.42 (m, 3H, Ar), 4.88 (s, 2H, CH2SO2), 3.85 (s, 3H, OCH3),
2.48 (m, 2H, COCH2CH2COOH), 2.43 (m, 2H, COCH2CH2COOH).
Low FAB (+)-MS, m/z 329.9 [M + H]+.
Hapten-5, ({({({[(4,6-dimethoxypyrimidin-2-yl)amino]carbonyl}-
amino)sulfonyl} methyl)benzoyl}amino)acetic acid. To a solution of
bensulfuron, the compound 2 (398 mg, 1 mmol) in dry N, N-
dimethylformamide (DMF) was added 1,1′-carbonyldiimidazole (CDI)
(172 mg, 1.06 mmol) at 0 °C. After the initial vigorous evolution of
gas, the reaction mixture (the temperature of which was allowed to
increase to room temperature) was stirred until no more gas evolved.
Solid ethyl glycinate hydrochloride (142 mg, 1.02 mmol) was added,
and the mixture was stirred until a clear solution resulted. On adding
water dropwise to this solution, a precipitate formed. The precipitate
was filtered out and the filtrate was concentrated under reduced pressure
to afford a pale-yellow solid. It was column chromatographed on silica
gel using ethyl acetate/n-hexane mixture (1:2, v/v) as an eluent to afford
To 1 mL of trifluoroacetic acid was added the compound 15 (150
mg, 0.49 mmol). The mixture was stirred at room temperature for 2 h
and then evaporated to dryness. Water (30 mL) was added to the
residue, and the mixture was extracted with ethyl acetate (2 × 30 mL).
The combined organic layer was dried over anhydrous magnesium
sulfate and concentrated on a rotary evaporator. The residue was purified
by using a chromatotron using ethyl acetate/n-hexane mixture (1:1, v/v)
as an eluent to give 113 mg of a transparent oily compound 16: TLC
1
compound 20 (213 mg, 74.9%): mp 165 °C. H NMR (CDCl3): δ
9.30 (br s, 1 H), 7.17 (br s, 1 H), 5.70 (s, 1 H), 4.26-4.18 (m, 4 H),
3.94 (s, 6 H), 1.08 (t, J ) 7.10 Hz, 3 H). 13C NMR (75.47 MHz,
CDCl3): δ 179.39, 168.21, 154.75, 151.23, 67.34, 54.34, 51.20, 37.91,
14.68. IR (vmax, KBr) 1723, 1610, 1454.
1
(methylene chloride/methanol, 1:9, v/v) Rf, 0.8. H NMR (CDCl3) δ
7.91 (d, 1H, J ) 7.5 Hz, Ar), 7.52 (m, 2H, Ar), 7.42 (m, 1H, Ar), 4.88
(s, 2H, CH2SO2), 4.85 (s, 2H, CH2SO2NH2), 4.32 (t, 2H, J ) 7 Hz,
OCH2CH2CH2CH2CH2COOH), 2.36 (m, 2H, OCH2CH2CH2CH2CH2-
COOH), 1.78 (m, 2H, OCH2CH2CH2CH2CH2COOH), 1.67 (m, 2H,
OCH2CH2CH2 CH2CH2COOH), 1.48 (m, 2H, OCH2CH2CH2CH2CH2-
COOH).
A solution of the compound 20 (103 mg, 0.29 mmol) in THF (5
mL) was cooled to 5 °C. To this solution was slowly added lithium
hydroxide monohydrate (36 mg, 0.86 mmol) in water (0.9 mL). The
resulting solution was stirred at 5-10 °C for 1.5 h and then diluted
with 1 N hydrochloric acid (0.86 mL). The mixture was concentrated
under reduced pressure to give the crude product. It was chromato-
graphed on silica gel using methanol/chloroform mixture (1:3, v/v) as
an eluent to afford hapten-5 (32 mg, 35%) as a pale-yellow crystalline
To a stirred solution of the compound 16 (113 mg, 0.37 mmol) in
4 mL of acetonitrile were added phenyl (4,6-dimethoxypyrimidin-2-
yl)carbamate (101 mg, 0.37 mmol) and DBU (113 mg, 0.74 mmol).
The reaction mixture was stirred at room temperature for 1 h, diluted
with 30 mL of ethyl acetate, and washed with 30 mL of water. The
organic layer was discarded, and the aqueous layer was acidified to
pH 3 with 5% hydrochloric acid, and then extracted with ethyl acetate
(2 × 30 mL). The combined organic layer was dried over anhydrous
magnesium sulfate, the solvent was removed by evaporation under
reduced pressure, and the residue was recrystallized from ethyl acetate/
n-hexane mixture to give 97 mg of compound 17 (hapten-3) as a white
solid: TLC (methanol/methylene chloride/acetic acid, 1:9:0.1, v/v/v)
Rf, 0.56. 1H NMR (DMSO-d6): δ 7.81 (d, 1H, J ) 7.5 Hz, Ar), 7.60-
7.40 (m, 3H, Ar), 5.92 (s, 1H, pyrimidine H), 5.29 (s, 2H, CH2SO2),
4.18 (m, 2H, OCH2CH2CH2CH2CH2COOH), 3.66 (s, 6H, OCH3), 2.21
(m, 2H, OCH2CH2CH2CH2CH2COOH), 1.65 (m, 2H, OCH2CH2CH2-
1
solid: mp 220 °C. H NMR (DMSO-d6): δ 9.30 (br s, 1H), 7.17 (br
s, 1H), 5.70 (s, 1H), 4.26-4.19 (m, 2H), 3.94 (s, 6H). 13C NMR (75.47
MHz, DMSO-d6): δ 171.87, 168.12, 154.75, 151.23, 69.73, 56.43,
38.76. IR (vmax, KBr) 3400-2460, 1714, 1456. Low FAB (+)-MS,
m/z 257 [M + H]+.
Hapten-6, 4-(3-(4,6-dimethoxypyrimidin-2-yl)-5-{[2-(methoxycar-
bonyl)benzyl] sulfonyl}-4-oxo-1,3,5-triazinan-1-yl)butanoic acid and
Hapten-7, 6-(3-(4,6-dimethoxypyrimidin-2-yl)-5-{[2-{methoxycarbonyl}-
benzyl]sulfonyl}-4-oxo-1,3,5-triazinan-1-yl)hexanoic acid. A mixture
of bensulfuron-methyl (2.1 g, 5.0 mmol), 4-aminobutyric acid (0.52 g,
5.0 mmol) for the synthesis of hapten-6 or 6-aminohexanoic acid (0.66
g, 5.0 mmol) for synthesis of hapten-7, and formaldehyde (35%, 5.6

1794 J. Agric. Food Chem., Vol. 50, No. 7, 2002
Lee et al.
g, 30.0 mmol) was stirred at 40 °C for 30 min in a three-necked round-
bottomed flask equipped with an internal thermometer and a Dean-
Stark trap. A solution of N-methylmorpholine (1.1 mL, 10.0 mmol),
1,4-dioxane (1 mL), and toluene (40 mL) was added, and the
temperature was raised to 85 °C, whereupon the solution became
homogeneous. Over a 90-min period, 35 mL of distillate was collected
in the trap, and toluene (15 mL) was added to the reaction mixture as
the internal temperature rose to 90-110 °C. The reaction mixture was
cooled and concentrated to a semisolid residue. Chromatography on
silica gel using methanol/chloroform mixture (1:10, v/v) as an eluent
afforded hapten-6 as a pale-yellow crystalline solid (1.57 g, 58.4%):
and 1 µg/mL and an antiserum dilution between 16000 and 256000.
Titers of the antisera produced by twelve rabbits were evaluated. Rabbits
A-C were immunized against hapten-1-KLH, D-F were immunized
against hapten-2-KLH, G-I were immunized against hapten-3-KLH,
and J-L were immunized against hapten-4-KLH. To check the titers
of the antisera by the homologous indirect ELISA, each antiserum was
diluted 256000-fold.
Indirect ELISA, Competitive Inhibition ELISA, and Cross-
Reactivities. Indirect ELISA and competitive indirect ELISA were
performed according to the method of Voller et al. (28) as modified
by Harrison et al. (29). For the checkerboard titration, an indirect ELISA
was conducted. That is, 96-well microtiter plates were coated with 100
µL/well of the hapten-BSA conjugate in a carbonate buffer and allowed
to stand overnight at 4 °C. On the following day, the plates were washed
five times with one-tenth strength PBST (0.1 × PBS with 0.05% Tween
20) and thoroughly tapped dry. Sites not coated with the conjugate
were blocked with 200 µL/well of 3% (w/v) skim milk with 1 × PBS.
After incubation at 37 °C for 1 h, the plates were washed as described
above. To the wells was added 100 µL of an anti-bensulfuron-methyl
antiserum diluted with 1 × PBS, and the plates were incubated at room
temperature for 1 h. After washing the plate, 100 µL of a secondary
antibody conjugated to the anti-rabbit IgG-horseradish peroxidase
diluted 1:10000 with 1 × PBST was added, and the plates were
incubated for 1 h at room temperature. The plate was washed, and 100
µL of a substrate solution (0.1 mL of 1% hydrogen peroxide and 0.4
mL of 0.6% 3,3′,5,5′-tetramethylbenzidine in dimethyl sulfoxide
(DMSO) added to 25 mL of citrate-acetate buffer, pH 5.5) was added
to each well. After 15 min at room temperature, the reaction was stopped
by adding 50 µL of 4 N sulfuric acid. The yellow-colored plate was
read spectrophotometrically in a dual wavelength mode at 450 nm using
a reference wavelength of 655 nm. The amount of the enzyme bound,
as indicated by the change of colorless substrate to blue product, is
directly related to the amount of the rabbit anti-hapten antibody bound
to the plate-coating antigen.
A competitive inhibition ELISA was used to assess the specificity
of the antibody to free bensulfuron-methyl and the cross-reactivities
of structurally related compounds to the antibody. For competition, 50
µL of standards in assay buffer or diluted samples were placed in the
wells, and 50 µL of the anti-bensulfuron-methyl antiserum diluted with
assay buffer was added to it. After these mixed for 30 s and incubated
at room temperature for 1 h, the plate was washed. The following
procedure was followed as described for the indirect ELISA. With the
inhibition ELISA format, analytes that do not react with the antibody
would produce absorbances near 100%; conversely, analytes that do
react with the antibody would decrease in percentage of absorbance.
Standard curves were calculated by mathematically fitting experimental
points to a four-parameter logistic equation (30) using a commercial
software package (Origin, Microcal).
Because the cross-reactivity is defined as the ability of structurally
related compounds of the target analyte to bind to the specific antibody
raised against the analyte, some other sulfonylurea herbicides, haptens,
and major metabolites of bensulfuron-methyl were tested for selectivity
of the ELISA by determining their respective IC50 values in the
competitive assays. Cross-reactivity values were calculated as the ratio
of the IC50 of the bensulfuron-methyl standard to the IC50 of the test
compounds and expressed as a percentage.
Water Samples. All water samples were collected from agricultural
areas where bensulfuron-methyl was not applied. Surface water samples
were collected from a pond, a stream, and a rice paddy.A groundwater
sample was from an underground well, and a soil leachate sample was
from a 1-m-deep lysimeter. The water samples were collected in brown
glass bottles and stored in a dark, cool place until the analyses. They
are characterized by pH, electric conductivity (EC), color, and smell,
as presented in Table 1.
ELISA and HPLC Analysis of Water Samples Fortified with
Bensulfuron-Methyl. ELISA. The samples were filtered through a 0.45-
µm filter to remove suspended solids. To test matrix effects, the water
samples were diluted 10 and 50 times with ultrapure water at pH 8.
For the recovery test, five concentrations (0, 2.5, 5, 10, and 25 ng/mL)
of bensulfuron-methyl in water samples were prepared with the
bensulfuron-methyl stock solution in ethyl acetate. For the ELISA of
1
mp 275 °C. H NMR (DMSO-d6): δ 12.02 (br s, 1H), 7.86 (d, J )
7.42 Hz, 1H), 7.50 (dd, J ) 7.43 Hz, J ) 1.87 Hz, 1H), 7.45 (dd, J )
7.36 Hz, J ) 1.87 Hz, 1H), 7.35 (d, J ) 7.36 Hz, 1H), 5.91 (s, 1H),
5.32 (s, 2H), 4.71 (s, 4H), 3.91 (s, 3H), 3.83 (s, 6H), 2.44-2.03 (m,
4H), 1.39-1.49 (m, 2H). 13C NMR (75.47 MHz, DMSO-d6): 180.04,
179.99, 168.61, 166.74, 158.78, 141.49, 136.25, 133.67, 126.40, 77.92,
58.11, 57.51, 57.28, 57.62, 54.43, 51.60, 30.75, 23.35. IR (vmax, KBr):
3300-2400, 1713, 1459. Low FAB (+)-MS, m/z 538 [M + H]+.
According to the method mentioned above, hapten-7 was also
obtained as the same colored crystalline solid (1.52 g, 53.7%): mp
1
250 °C; H NMR (DMSO-d6) δ 8.55 (br s, 1H), 7.69-7.58 (m, 1H),
7.51-7.33 (m, 3H), 5.83 (s, 1H), 5.30 (s, 2H), 4.72 (s, 4H), 3.92 (s,
3H), 3.86 (s, 6H), 2.46-2.05 (m, 4H), 1.47-1.36 (m, 6H); 13C NMR
(75.47 MHz, DMSO-d6) δ 183.08, 180.01, 168.16, 165.58, 158.77,
141.61, 136.56, 131.34, 129.05, 77.25, 58.56, 57.49, 57.28, 56.70, 54.69,
51.10, 41.68, 35.41, 27.56, 23.45; IR (νmax, KBr) 3300-2400, 1714,
1463; Low FAB (+)-MS, m/z 566 [M+H]+.
Conjugation of Carboxylic Acid Haptens to Carrier Proteins.
The haptens were coupled covalently to the lysine groups of the carrier
proteins such as keyhole limpet hemocyanin (KLH) and bovine serum
albumin (BSA) according to the activated ester method (23). That is,
each hapten (0.04 mmol) was dissolved in 0.2 mL of dry DMF with
equimolar N-hydroxysuccinimide (NHS) and a 10% molar excess of
dicyclohexylcarbodiimide. After stirring at 22 °C for 5 h, the
precipitated dicyclohexylurea was removed by filtration, and about 0.2
mL of the active ester was added slowly to the protein solution (10
mg of protein in 1 mL of 0.05 M borate buffer at pH 8) with vigorous
stirring. The reaction mixture was stirred gently at 4 °C for 24 h to
complete the conjugation and then dialyzed exhaustively against normal
strength phosphate-buffered saline (1 × PBS; 8 g of NaCl, 0.2 g of
KH2PO4, 1.2 g of Na2HPO4, and 0.2 g of KCl), which was changed
twice a day for 5 days. Finally, the conjugates were dispensed into the
2-mL cryogenic vial and stored at -80 °C.
To calculate the amount of the hapten-protein conjugate to be used
as immunogens or coating antigens as a protein equivalent, its protein
content was determined by the Bio-Rad protein assay based on the
method of Bradford (24). The extent of coupling of each hapten to the
carrier protein was determined according to the UV spectrophotometric
(25) or the trinitrobenzenesulfonic acid (TNBSA) method (26).
Immunization. Female New Zealand white rabbits weighing 3 kg
were used for raising polyclonal antibodies. Routinely, 100 µg (protein
equivalent) of each immunogen (hapten-KLH conjugate) in 0.5 mL
of 0.85% saline was thoroughly emulsified with an equal volume of
Freund’s adjuvant. The emulsion was subcutaneously injected at five
different sites on the neck and back of a rabbit. Complete Freund’s
adjuvant was used in the first injection, and Freund’s incomplete
adjuvant was used for the subsequent boost injections. Boosts were
given every three weeks in the same manner. On the seventh day after
each boost, about 10 mL of blood sample was drawn from the jugular
vein of the ear to check the titer of the polyclonal antibody. The blood
sample was allowed to coagulate for about 2 h at room temperature,
and then it was left to stand overnight in a refrigerator. The serum was
decanted and centrifuged (800g), and then the supernatant was stored
in conveniently sized aliquots at -80 °C. Boosts were given six times.
Checkerboard Titration. A checkerboard titration (27) was per-
formed with each of the antisera collected from each rabbit. The
checkerboard assay selected the combination of antiserum dilution and
coating antigen concentration (hapten-BSA conjugate) that would
provide the greatest sensitivity in ELISA. The optimized ELISA for
bensulfuron-methyl used a coating antigen concentration between 0.01

ELISA for Bensulfuron-Methyl
J. Agric. Food Chem., Vol. 50, No. 7, 2002 1795
methyl was determined by HPLC (Hewlett-Packard HP 1100 series)
equipped with a diode array detector on a LiChrosorb R-18 (250 mm
× 4.6 mm i.d.). Operating conditions were as follows: injection volume,
20 µL; flow rate, 1.0 mL/min; detection wavelength, 254 nm; mobile
phase, acetonitrile/water (pH 2.3 adjusted with H3PO4) (50:50, v/v).
Table 1. Characteristics of Water Samples Collected from the
Agricultural Areas near Cheongju
a
water source
pH
EC
color
smell
odorless
slightly rotten odor
odorless
odorless
odorless
pond
stream
rice paddy
soil leachate
underground
1 × PBS
7.8
6.7
8.4
7.9
7.2
8.0
0.1
0.2
0.3
0.4
0.3
pale yellow
pale yellow
pale yellow
pale yellow
colorless
RESULTS AND DISCUSSION
Synthesis of Haptens and Their Conjugation to Carrier
Proteins. The herbicide bensulfuron-methyl in itself cannot be
used as an immunogen because of its low molecular weight
(MW 410.4). Therefore, haptens mimicking the analyte herbicide
and containing reactive groups for conjugation to carrier proteins
should be synthesized to develop an ELISA for it. The hapten
is also a small molecule with a great diversity of reactive groups
such as carboxyl, amino, or hydroxyl group for conjugation to
proteins, and cannot, by itself, elicit an antibody response. In
the present study, seven analogues of the target analyte were
synthesized for use as immunogens and plate-coating antigens.
For the production of antibodies capable of recognizing ben-
sulfuron-methyl, the immunizing haptens were designed as
almost perfect mimics of the structure of the target molecule in
terms of electronic and hydrophobic properties. The immunizing
hapten-1 was synthesized as bensulfuron with a carboxyl group
by hydrolyzing the aromatic methyl ester group in the target
molecule. Because the spacer arms with different lengths to
connect the target analyte to the carrier protein have an effect
on the formation of the specific and sensitive antibody (31-
32), two other immunizing haptens, hapten-2 and hapten-3, were
synthesized by replacing the methyl group in the ester of the
13.2
colorless
odorless
a Electric conductivity, reported as mS/cm.
bensulfuron-methyl, the aliquots of each sample were diluted 50 times
with ultrapure water of pH 8. Each analysis was done in triplicate.
HPLC. One liter of each groundwater sample fortified with the
analyte by the above procedures was transferred into a 2-L separatory
funnel, and 50 mL of a saturated NaCl solution was added to it. The
solution was acidified to pH 3 with 4 N hydrochloric acid and then
extracted twice with 80 mL of methylene chloride. The combined
organic layer was dried over anhydrous sodium sulfate, and the solvent
was removed by evaporation. After the residue was redissolved in 10
mL of distilled water, the pH of the sample was adjusted to 2-3 with
4 N hydrochloric acid. A 6-mL cartridge packed with 0.5-g C18 (Alltech
Associates, Inc.) was preconditioned by passing 5 mL of methanol
through it, followed by 5 mL of water. A low vacuum and a flow rate
of 5-10 mL/min were used. The acidified sample and then 5 mL of
distilled water were passed through the cartridge. The eluate was
discarded and the cartridge was dried by suction for 15 min. Finally,
the cartridge was eluted with 5 mL of 0.1% (v/v) acetic acid in ethyl
acetate and the eluate was evaporated to dryness under reduced pressure.
The residue was redissolved in 2.5 mL of acetonitrile, and bensulfuron-
Figure 1. Synthetic route of hapten-2.

1796 J. Agric. Food Chem., Vol. 50, No. 7, 2002
Lee et al.
Figure 2. Synthetic route of hapten-3.
of the immunizing haptens were identical to the target molecule
in structure and geometry (data not shown).
Heterology is commonly used to eliminate problems coming
from no or poor inhibition by the target compound, associated
with the strong affinity of the antibodies for the bridging groups.
It usually results in somewhat weaker recognition of plate-
coating antigens than recognition of the target analyte. Thus,
lower analyte concentrations can compete with these reagents
under equilibrium conditions, which results in better assay
sensitivity than homologous system (33-34). For the heterolo-
gous bensulfuron-methyl ELISA in this study, heterology
included hapten heterology using different hapten structures,
site heterology, linker heterology using spacer arms with
different lengths (35-36), and carrier heterology. Additional
haptens for plate-coating were synthesized (Figures 4 and 5):
one coating hapten (hapten-5) contained a partial structure of
the target molecule and two other coating haptens (hapten-6
and -7) contained a cyclization of the sulfonylurea bridge in
the target molecule. All of the seven haptens containing spacer
arms of different lengths and partial or cyclized structures of
the sulfonylurea bridge of the target molecule were conjugated
to BSA for use as coating antigens to compare homologous and
heterologous ELISA formats.
Figure 3. Synthetic route of hapten-4.
target molecule with four- and six-carbon spacer arms with a
carboxyl group at the end for conjugation (Figures 1 and 2).
In contrast to the above three haptens, hapten-4 was a mimic
of only the aryl sulfonamide moiety of the analyte molecule,
the other part of which was replaced by a three-carbon spacer
arm. It was synthesized by the reaction of succinic anhydride
as a spacer arm with the methylaminosulfonyl-o-toluate moiety
of the analyte (Figure 3). Hapten-4 was designed adopting the
methods by Schlaeppi et al. (12) and Simon et al. (14). Using
haptens that were the sulfonamide moieties of triasulfuron and
metsulfuron-methyl containing succinic acid as a spacer arm,
they produced antibodies with a higher affinity for the parent
herbicides than a hapten imitating the complete analyte mol-
ecule.
The protein contents of the hapten-protein conjugates were
in the range of 3.8-6.9 mg/mL. By the UV spectrophotometric
method, the conjugation molar ratios of each hapten to BSA
were determined as 26/1, 21/1, 59/1, 34/1, and 79/1 for hapten-
1, -2, -3, -5, and -7, respectively, and those of each hapten to
KLH were 1677/1, 606/1, and 2918/1 for hapten-1, -2, and -3,
respectively. On the other hand, the main peak of hapten-4 or
The four haptens were conjugated to KLH for use as
immunogens for the antibody production. The molecular models

ELISA for Bensulfuron-Methyl
J. Agric. Food Chem., Vol. 50, No. 7, 2002 1797
coating antigens and antisera were screened via the inhibition
by two different concentrations (10 and 1000 ng/mL) of the
analyte dissolved in the assay buffer, using the homologous or
heterologous competitive indirect ELISA system. The inhibition
ratio was expressed as a percentage of the difference between
the absorbance of the analyte-free buffer and that of the analyte-
containing buffer, divided by the former. The antibodies raised
against hapten-2, -3, and -4 with a spacer arm of a four- or
six-carbon chain were more specific to the target analyte in the
heterologous ELISAs than those raised against hapten-1 without
a bridge group. These results prove that a spacer arm in a hapten
is essential for the production of antibodies specific to the
analyte.
There was almost no or very low inhibition by the analyte in
homologous ELISAs using the same hapten for a coating antigen
and an immunogen. Whereas, there were very high inhibitions
in heterologous ELISAs using the combinations of hapten-1,
-5, -6, and -7 as coating antigens with the antisera raised against
the hapten-2-KLH and hapten-3-KLH immunogens, and the
combinations of hapten-1, -2, -3, -5, -6, and -7 as coating
antigens with the antisera against the hapten-4-KLH immu-
nogen. It is interesting that rabbits injected with hapten-2 and
hapten-3 closely related to the analyte bensulfuron-methyl
produced antisera exhibiting a high inhibition, whereas rabbits
injected with hapten-4 mimicking only a portion of the analyte
structure also produced antisera showing a similar inhibition.
Figure 4. Synthetic route of hapten-5.
The nine heterologous ELISAs using the combinations of the
coating hapten-1, -2, -3, -5, and -7 and the antiserum D, F, and
K, which showed higher inhibition ratios, were conducted at
10 concentrations of bensulfuron-methyl. The IC50 values of
the rabbit antiserum D and F raised against the hapten-2-KLH
immunogen were, respectively, 0.47 and 0.70 ng/mL on a plate
coated with hapten-1-BSA, 0.68 and 4.98 ng/mL with hapten-
5-BSA, and 23 and 0.61 ng/mL with hapten-7-BSA, and those
of the antiserum K against the hapten-4-KLH were 0.16 ng/
mL with the hapten-1-BSA, 0.18 ng/mL with the hapten-2-
BSA, and 0.51 ng/mL with the hapten-3-BSA. For the
development of the bensulfuron-methyl ELISA, we optimized
three sensitive heterologous ciELISAs using three combinations
of the coating antigens with the antisera: assays I, II, and III
were the combinations of the coating hapten-1 with the
antiserum D, of the coating hapten-2 with the antiserum K, and
of the coating hapten-7 with the antiserum F, respectively. The
assay I was used to optimize blocking conditions of the coated
plate and the buffer for the assay, because the antiserum D had
the highest titer.
Figure 5. Synthetic routes of hapten-6 and hapten-7.
hapten-6 was overlapped with that of BSA or KLH. Therefore,
the conjugation ratios of the hapten-4-protein conjugates and
the hapten-6-BSA conjugate were determined by the TNBSA
method. The coupling density was estimated by comparing the
absorbance with the corresponding values of hapten-free
proteins. From the available amino groups, 38% of hapten-4
and 64% of hapten-6 were conjugated in the hapten-BSA
conjugates and 47% of hapten-4 was conjugated in the hapten-
KLH conjugate.
Titers of the Antisera. The four hapten-KLH conjugates
were injected seven times into each rabbit as immunogens,
respectively. The antisera collected after each boost were
subjected to titration by the homologous indirect ELISA. All
of the antisera showed higher titers after the fourth, fifth, or
sixth boosting than those of the others. These results indicate
that specific antibodies in the rabbit antiserum were produced
against each immunogen. The final antisera were used for the
subsequent screening in search of antibodies specific to the target
compound bensulfuron-methyl. All antisera did not show any
significant affinity for BSA itself as a coating antigen.
Screening and Selection of Coating Antigens and Antisera.
To establish a sensitive ELISA, all combinations between
Optimization of the ELISA. To determine the bensulfuron-
methyl residues in the agricultural and environmental samples,
it is essential to develop an ELISA with optimum sensitivity.
For that purpose, the effects of blocking agents on the plate
coated with a coating antigen, and the assay buffer-related
factors such as solvent, detergent, ionic strength, and pH were
evaluated.
Effect of Blocking Agents. To minimize the background and
maximize signal-to-noise ratios in the assay, blocking agents
were evaluated to hinder nonspecific sorption of an antibody
onto the less-coated plate. Because there is no universal blocking
buffer for all immunoassays, various blocking agents for each
immunoassay need to be tested. As shown in Figure 6, all the
tested blockers enhanced the assay sensitivity compared to no
blocking. Skim milk as a blocker showed a lower background
signal, a higher ratio of maximum absorbance (A) to minimum
absorbance (D), and a higher sensitivity for the ELISA than
other blockers. In particular, blocking with 3% skim milk at 37

1798 J. Agric. Food Chem., Vol. 50, No. 7, 2002
Lee et al.
Figure 6. Effect of the blocking agents on the sensitivity of the ELISA.
Table 2. Effect of the Assay Buffer-Related Factors on the Sensitivity
higher than that without the organic solvents, indicating that
an increase in the amount of organic solvent decreases the assay
sensitivity in the bensulfuron-methyl ELISA. The amounts of
the organic solvents that are less than 4% in the assay buffer
had little effect on the sensitivity of the ELISA. It will be
necessary to use a small amount of organic solvent in the assay
buffer to reduce assay variability by decreasing interference of
lipid micelles with complex matrixes and by reducing binding
of analyte to surfaces of the plate (37). As water solubility of
bensulfuron-methyl at pH 8 is 1200 mg/L (38), the use of large
amounts of organic solvents will not be necessary for the ELISA.
Meanwhile, Tween 20 is a nonionic detergent and has been
used in immunoassays to reduce nonspecific binding and
improve sensitivity (39). However, in this study, as shown in
Figure 7, the IC50 value in the buffer to which 0.05% (v/v)
Tween 20 was added was about twice higher than that without
the detergent, indicating that the addition of Tween 20 to the
buffer did not enhance the sensitivity of the ELISA for the
detection of bensulfuron-methyl. This result is in agreement with
the studies of the esfenvalerate ELISA (40) and the polychlo-
rinated dibenzo-p-dioxin ELISA (41) that showed Tween 20
decreased the sensitivity due to nonspecific hydrophobic interac-
tions between the detergent and nonpolar small analytes in an
aqueous environment, thereby interfering with the specific
analyte-antibody interaction. Manclu´s and Montoya (42) also
reported that Tween 20 had no effect on the sensitivity of an
ELISA for a highly polar compound such as TCP (3,5,6-
trichloro-2-pyridinol). The buffer without a detergent in the
ELISAs for the polar sulfonylurea herbicides, metsulfuron-
methyl and chlorsulfuron, might be also used for this reason
(14, 43).
a
of the ELISA
Amax
slope
IC50 (ng/mL)
Amin
factor
(A)
(B)
(C)
(D)
A/D
blocking temperature
rt
1.342
1.055
1.34
1.46
0.31
0.17
0.048
0.030
27.96
35.17
37 °C
organic solvent in the assay bufferb
methanol (%)
0
0.994
0.996
0.939
0.778
1.36
1.05
0.83
0.96
0.20
0.30
0.34
0.63
0.024
0.004
−0.011
0.020
2
4
10
acetone (%)
0
0.945
0.992
0.955
0.957
0.994
0.85
0.72
0.77
0.63
0.90
0.12
0.12
0.20
0.32
0.47
−0.008
−0.019
−0.037
−0.069
−0.017
2
4
10
20
concentration of the assay buffer (ionic strength)c
0.5
1
1.5
1.055
0.668
0.377
1.46
1.35
1.00
0.17
0.14
0.15
0.030
0.015
0.003
dilution of the antibody
1:32000
1:48000
1:64000
1.080
1.054
0.941
1.41
1.46
1.23
0.22
0.17
0.10
0.022
0.030
0.006
a Assay conditions: coating antigen, hapten-1−BSA (1 µg/mL); blocking with
3% skim milk in 1 × PBS at 37 °C; antiserum, rabbit D (1:48000) in 1 × PBS;
standard series of bensulfuron-methyl were dissolved in ultrapure water; goat anti-
rabbit IgG-HRP (1:10000). Data are the means of quadruplicate samples.
b Antiserum, rabbit D (1:32000). c The ionic strength is the mean of the buffers
containing the analyte and the antiserum.
°C resulted in lower nonspecific binding of an antibody to the
solid phase, and hence the sensitivity almost doubled that at
room temperature (Table 2).
Effect of Organic SolVent and Detergent. The effects of the
organic solvent (methanol or acetone) and detergent on the
ELISA were evaluated. As shown in Table 2, the assay
sensitivity was higher in the buffer containing no or a small
amount of organic solvents. The IC50 value in the assay buffer
containing 10% methanol or acetone was about three times
Effect of pH and Ionic Strength. To determine the effect of
pH on the assay, the phosphate buffer was used in the range of
pH 6.5 to 9.5, and results were compared with one another. As
shown in Figure 8, in the range tested, the pH value affected
the assay. The sensitivity of the ELISA was a little higher under
slightly alkaline conditions than under slightly acidic and neutral
conditions, as shown by the IC50 values. This means that the
interaction between the antibody and the target analyte was most
favored under this alkaline condition of pH 8.5. Because

ELISA for Bensulfuron-Methyl
J. Agric. Food Chem., Vol. 50, No. 7, 2002 1799
Figure 7. Effect of the detergent in the assay buffer on the sensitivity of the ELISA.
Figure 8. Effect of pH on the sensitivity of the ELISA, as shown by the IC50 value.
bensulfuron-methyl is most stable under a slightly alkaline
condition (pH 8), the optimum pH range of the assay buffer
was set at 8-8.5 for the ELISA.
of the antibody. It is possible to obtain a more sensitive ELISA
for bensulfuron-methyl by decreasing the concentration of the
antibody.
As shown in Table 2, the binding of the antibody to the
coating antigen decreased with the increasing salt concentration
of the assay buffer. The optimum ion strength of the assay
phosphate buffer for a maximum binding was that of 0.5 ×
PBS.
On the basis of these results, the optimal conditions for the
bensulfuron-methyl ELISA are summarized as follows (Table
3): A quantity of 1 µg/mL of the hapten-1-BSA conjugate as
a coating antigen was coated onto the plate and stored at 4 °C
overnight, and then the plate was blocked with 3% skim milk
at 37 °C for 1 h. The antiserum D raised against the hapten-
2-KLH conjugate as an immunogen was diluted 64000-fold
in the assay buffer at pH 8 without detergent or organic solvent
and competed with the target analyte dissolved in pH 8 ultrapure
water in the blocked plate. With the heterologous assay I
optimized for bensulfuron-methyl under these conditions, the
IC50 value of the analyte as low as 0.17 ( 0.04 ng/mL was
obtained, showing the detection range of 0.04-0.60 ng/mL, and
Dilution of the Antisera. The concentrations of immu-
noreagents such as the antisera and the coating antigens selected
for the ELISA were determined in the checkerboard titration.
In addition, an intensive investigation for a more sensitive assay
was also carried out at different concentrations of the antiserum
on the same amount of a coating antigen. As shown in Table
2, the higher the antibody dilution is, the lower the IC50 value
is. The ELISA becomes more sensitive by reducing the amount

1800 J. Agric. Food Chem., Vol. 50, No. 7, 2002
Table 3. Optimized Conditions for the ELISAs
Lee et al.
Kawada et al. (15) reported on the monoclonal antibody-based
ELISAs for bensulfuron-methyl, using haptens with a spacer
arm of various carbon chains. The detection ranges were
reported to be 0.2-2 and 0.5-5 ng/mL in the competitive
indirect and direct inhibition ELISAs, respectively. Whereas in
our ELISA, the detection range was 0.04-0.60 ng/mL in the
assay I, which is far lower than their detection range. Besides,
the haptenic synthesis was quite different from theirs. Mean-
while, the monoclonal antibody-based fluoroimmunoassay for
bensulfuron-methyl developed by Yuan et al. (17) showed the
detection limit of 0.024 ng/mL. Even if they used a tetradentate
â-diketonate europium fluorescent chelate to label a bensulfuron-
methyl BSA conjugate for this assay, the detection limit was
almost the same as ours, which was 0.03 ng/mL in the assay I.
coating
antigen
blocking antiserum assay buffer
assay immunogen
agent
3% skim milk rabbit D
hapten-1−BSA at in 1 × PBS (1:64000)
(dilution)
(pH 8)
I
hapten-2−KLH 1 µg/mL of
0.5 × PBS
4 °C overnight
at 37 °C
II
hapten-4−KLH 0.5 µg/mL of
3% skim milk rabbit K
1 × PBS
1 × PBS
hapten-2−BSA at in 1 × PBS (1:30000)
37 °Cfor 2h
III hapten-2−KLH 1 µg/mL of
at 37 °C
3% skim milk rabbit F
hapten-7−BSA at in 1 × PBS (1:24000)
37 °Cfor 2h at 37 °C
the lowest detection limit (LOD) of 0.02 ng/mL (Figure 9).
Whereas, as shown in Table 3 and Figure 9, under the
optimized conditions of the assays II and III, the IC50 values
of 0.09 ( 0.02, and 0.09 ( 0.03 ng/mL were obtained by using
hapten-2-BSA and hapten-7-BSA, respectively, as plate-
coating antigens and hapten-4-KLH and hapten-2-KLH
conjugates as immunogens, showing the detection ranges of
0.01-0.60 and 0.04-0.25 and LODs of 0.002 and 0.03 ng/
mL, respectively.
Cross-Reactivities. In assay I that is the heterologous
combination of hapten-2-KLH (immunogen) and hapten-1-
BSA (coating antigen), the antiserum D recognized some
structurally related compounds, but not the two possible
metabolites. On the basis of the cross-reactivity (Table 4), the
antigenic determinants in each compound to which the rabbit
D antibody binds specifically would be a phenyl group
connected to the sulfonylurea moiety via methylene, oxygen,
Figure 9. Standard curves obtained under the optimized conditions in the ELISAs.

ELISA for Bensulfuron-Methyl
J. Agric. Food Chem., Vol. 50, No. 7, 2002 1801
Table 4. Cross-Reactivity of Some Structurally Related Compounds to the Rabbit D and K Antisera in the ELISAsa
aRabbit D and K antisera were raised against the immunogens hapten-2−KLH conjugate (assay I) and hapten-4−KLH conjugate (assay II), respectively. b % Cross-
reactivity (CR) ) (IC50 of bensulfuron-methyl/IC50 of other compounds) × 100. c NI, no inhibition.
or NH moieties. The fact that the covalent radii of carbon,
oxygen, and nitrogen atoms are 0.771, 0.74, and 0.74 Å,
respectively, indicates that the steric fitness is very important
in the antigen-antibody interaction. This argument is based on

1802 J. Agric. Food Chem., Vol. 50, No. 7, 2002
Lee et al.
Table 5. Recovery of the Analyte Bensulfuron-Methyl Fortified to
the result that the sulfonylureas such as azimsulfuron, imazo-
sulfuron, pyrazosulfuron-ethyl, and nicosulfuron that contain 4,6-
dimethoxypyrimidine and sulfonylurea moieties without meth-
ylene group showed very low cross-reactivities. The fact that
metabolite-1 and metabolite-2 containing no complete sulfo-
nylurea moiety showed no cross-reactivity supports the above
reasoning.
Some Water Samples by the ELISA
50-fold
diluted
(ng/mL) (ng/mL)
mean
recovery
(%, n ) 3)
coefficient of
variation
(%)a
water
sample
fortified
detected
(ng/mL)
b
rice paddy
0
2.5
5
0
<LOD
-
84
108
83
94
-
108
86
81
106
-
92
90
100
122
-
92
94
117
120
-
0.4
4
0.05
0.10
0.20
0.50
0
0.042
0.108
0.166
0.470
<LOD
0.054
0.086
0.162
0.530
<LOD
0.046
0.090
0.200
0.612
<LOD
0.046
0.094
0.234
0.600
<LOD
0.058
0.082
0.250
0.482
Schlaeppi et al. (12) reported an ELISA for the sulfonylurea
herbicide triasulfuron based on monoclonal antibodies (MAbs).
They generated MAbs with high affinity for the analyte by a
simple hapten corresponding only to the chloroethoxy sulfona-
mide moiety of triasulfuron with an additional succinic acid
spacer. They indicated that the urea function of the bridge
structure was a prerequisite for the binding of the MAb. In
addition, they concluded that the chloroethoxy group interacted
with the MAb. Similarly, Kelley et al. (11) worked on another
sulfonylurea herbicide, chlorsulfuron. They suggested that the
bridge and heterocycle structure is related to the antigen-
antibody binding, based on the cross-reactivity changes as
affected by the modification of the above structures. In the case
of assay II, in which the combination of hapten-4-KLH
(immunogen) and hapten-2-BSA (coating antigen) was used,
only ethoxysulfuron exhibited a low cross-reactivity (2.8%)
except for hapten-4 that was used as the immunogen. The cross-
reactivities of the two compounds would be based on the fact
that ethoxysulfuron is very similar to bensulfuron-methyl in
structure in that the lengths of the -COOCH3 in bensulfuron-
methyl and -OCH2CH3 group in ethoxysulfuron are both 5.14
Å, in addition to the similar sizes of the -CH2- group in the
former and the oxygen atom in the latter. Meanwhile, in assay
III (not presented), where the combination of hapten-2-KLH
(immunogen) and hapten-7-BSA (coating antigen) was used,
quite a few sulfonylurea compounds showed relatively high
cross-reactivities. This result could be due to the fact that the
structure of coating antigen is quite different from that of the
immunogen, rendering high affinity between the antibody and
the test compound.
8
10
25
0
5
5
pond
2
2.5
0.05
0.10
0.20
0.50
0
0.05
0.10
0.20
0.50
0
0.05
0.10
0.20
0.50
0
0.05
0.10
0.20
0.50
4
5
10
25
0
10
3
7
stream
2
2.5
1
5
10
25
0
5
8
10
1
underground
soil leachate
2.5
2
5
10
25
0
6
6
9
5
2.5
116
82
125
96
2
5
10
25
1
3
8
a Coefficient of variation is defined as the standard deviation divided by the
mean, expressed as a percentage. b LOD means the lowest detection limit (0.03
ng/mL) in assay I.
was adopted for the better quantitation of the analyte in various
water samples.
To validate the ELISA, the same fortified groundwater
samples were subjected to HPLC after a cleanup step. The
equation of the linear regression correlation between the ELISA
(Y) and HPLC (X) results was Y ) 1.38X - 1.78 (r ) 0.999, n
) 15). The ELISA is comparable to HPLC in the analysis of
bensulfuron-methyl residues in water samples. Therefore, this
ELISA could be used as an alternative to the conventional
instrumental methods for monitoring bensulfuron-methyl resi-
dues in agricultural and environmental samples.
Matrix Effect. Because of the matrixes such as inorganic
and organic compounds contained in water samples, the test
samples used in ELISA should be diluted or purified by a solid-
phase extraction or other methods prior to the determination of
bensulfuron-methyl residues. In assay I, the water samples
collected from nearby agricultural areas were diluted 10 and
50 times with ultrapure water, after filtration and pH adjustment
to 8, to prevent the interference by matrixes. Standard curves
of the water samples were prepared with each diluted water
sample, and compared to the calibration curve prepared with
ultrapure as a control. The matrix effects of water samples by
the 50-fold dilution fairly diminished, showing that their
standard curves were almost close to the control curve (data
not shown). These results indicate that the ELISA can determine
bensulfuron-methyl residues in water samples at the ppb-level
only by simple dilution.
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