Development of an enzyme-linked immunosorbent assay for the organophosphorus insecticide bromophos-ethyl

Article abstract of DOI:10.1021/jf025703+

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the organophosphorus insecticide bromophos-ethyl. Four bromophos-ethyl derivatives (haptens) were synthesized and were coupled to carrier proteins through the pesticide thiophosphate group to use as an immunogen or as a coating antigen. Rabbits were immunized with either one of two haptens coupled to bovine serum albumin for production of polyclonal antibodies, and the sera were screened against one of the haptens coupled to ovalbumin. Using the serum with highest titer, an antigen-coated ELISA was developed, which showed an IC₅₀ of 3.9 ng/mL with a detection limit of 0.3 ng/mL (20% inhibition). An antibody-coated ELISA using an enzyme tracer was also developed, which showed an IC₅₀ of 6.5 ng/mL with a detection limit of 1.0 ng/mL (20% inhibition). The antibodies showed negligible cross-reactivity with other organophosphorus pesticides except with the insecticides bromophos-methyl and chlorpyrifos in the antibody-coated assay only. Recoveries of bromophosethyl from fortified crop and water samples ranged from 82 to 128% and from 95 to 127%, respectively.

Full text of DOI:10.1021/jf025703+

J. Agric. Food Chem. 2002, 50, 6675−6682 6675
Development of an Enzyme-Linked Immunosorbent Assay for
the Organophosphorus Insecticide Bromophos-ethyl
KWANG-OK KIM,† YOO JUNG KIM,‡ YONG TAE LEE,‡ BRUCE D. HAMMOCK,§ AND
HYE-SUNG LEE*,†
Department of Food Science and Nutrition, Kyungpook National University, Daegu 702-701, Korea;
Department of Biochemistry and Institute of Natural Sciences, Yeungnam University,
Kyongsan 712-749, Korea; and Department of Entomology and Cancer Research Center,
University of California, Davis, California 95616
A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative
detection of the organophosphorus insecticide bromophos-ethyl. Four bromophos-ethyl derivatives
(haptens) were synthesized and were coupled to carrier proteins through the pesticide thiophosphate
group to use as an immunogen or as a coating antigen. Rabbits were immunized with either one of
two haptens coupled to bovine serum albumin for production of polyclonal antibodies, and the sera
were screened against one of the haptens coupled to ovalbumin. Using the serum with highest titer,
an antigen-coated ELISA was developed, which showed an IC50 of 3.9 ng/mL with a detection limit
of 0.3 ng/mL (20% inhibition). An antibody-coated ELISA using an enzyme tracer was also developed,
which showed an IC50 of 6.5 ng/mL with a detection limit of 1.0 ng/mL (20% inhibition). The antibodies
showed negligible cross-reactivity with other organophosphorus pesticides except with the insecticides
bromophos-methyl and chlorpyrifos in the antibody-coated assay only. Recoveries of bromophos-
ethyl from fortified crop and water samples ranged from 82 to 128% and from 95 to 127%, respectively.
KEYWORDS: Bromophos-ethyl; insecticide; immunoassay; ELISA
INTRODUCTION
(5, 6). An enzyme-linked immunosorbent assay (ELISA) for
this pesticde has not yet been reported.
Due to the widespread use of pesticides, there is increasing
concern over food and environmental contamination caused by
their use. The current methods such as gas chromatography (GC)
and high-performance liquid chromatography (HPLC) have been
used successfully, with high sensitivity and reliability, for
analysis of many pesticides (1). However, these classical
methods require a high capital expenditure and skilled analysts
and involve time-consuming sample preparation steps. There-
fore, there is a growing demand for more rapid and economical
methods for determining pesticide residues. Immunoassays have
recently emerged as an alternative to the traditional methods
that can meet such demands. Immunochemical techniques that
have been used extensively in clinical laboratories began recently
to gain acceptance as a fast, sensitive, and cost-effective tools
for detecting trace amounts of chemicals such as pesticides (2).
Bromophos-ethyl [O,O-diethyl O-(4-bromo-2,5-dichloro-
phenyl)phosphorothioate] is an organophosphorus insecticide
and acaricide, which is effective against a wide range of insects
(3). The most sensitive and toxicologically relevant effect after
administration of bromophos-ethyl is inhibition of acetylcho-
linesterase activity (4). Analysis of bromophos-ethyl is carried
out by multiresidue methods using gas-liquid chromatography
The development of an immunoassay requires the production
of antibodies to the analyte. Because pesticides are small
molecules, pesticide derivatives, namely haptens, must be
synthesized and coupled to carrier proteins to induce antibody
production. One type of hapten for organophosphorus pesticides
is the one with an amino carboxylic acid bridge at thiophosphate
group, which has been used successfully in the development of
ELISA for several organophosphorus pesticides (7-12). We
have developed a novel method for the synthesis of such
haptens, which is much easier than the previous one (7). This
paper describes the application of this method to the synthesis
of haptens for bromophos-ethyl from which specific antibodies
to bromophos-ethyl were obtained. Using the antibodies, sensi-
tive and selective ELISAs for bromophos-ethyl were developed.
MATERIALS AND METHODS
Chemicals and Instruments. Organophosphorus pesticides includ-
ing bromophos-ethyl were purchased from Dr. Ehrenstorfer (Augsburg,
Germany). Ethyl dichlorothiophosphate, 4-aminobutyric acid, 5-ami-
novaleric acid, 6-aminocaproic acid, 3-(methylamino)butyric acid
hydrochloride, N-hydroxysuccinimide, 1,3-dicylohexylcarbodiimide,
4-(dimethylamino)pyridine, chlorform-d, silica gel (60-230 mesh), and
polyoxyethylene (20) sorbitan monolaurate (Tween 20) were obtained
from Aldrich (Milwaukee, WI). Bovine serum albumin (BSA), oval-
bumin (OVA), peroxidase-labeled goat anti-rabbit IgG, Freund’s
† Kyungpook National University.
‡ Yeungnam University.
§ University of California.
10.1021/jf025703+ CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/04/2002

6676 J. Agric. Food Chem., Vol. 50, No. 23, 2002
Kim et al.
acetate/acetic acid (19:9:1)] of the residue gave 72 mg (33%) of a white
solid: 1H NMR (CDCl3) δ 7.67 (1H, d, J ) 0.7, ar), 7.64 (1H, d, J )
1.6, ar), 4.21 (2H, dxqxd, J ) 9.2, 7.1, and 1.8, CH2CH3), 3.39 (1H,
qn, J ) 7.1, NH), 3.19 (2H, dxq, J ) 12 and 6.8, NHCH2), 2.46 (2H,
t, J ) 7.2, CH2CO2H), 1.88 (2H, qn, J ) 7.0, CH2CH2CH2), 1.36 (3H,
t, J ) 7.1, CH2CH3).
Haptens B, C, and D were synthesized according to the same
procedure as for hapten A, using 5-aminovaleric acid, 6-aminocaproic
acid, and 4-(N-methylamino)butyric acid, respectively.
Hapten B. The yield was 40%: 1H NMR (CDCl3) δ 7.67 (1H, d, J
) 0.6, ar), 7.65 (1H, d, J ) 1.1, ar), 4.21 (2H, m, CH2CH3), 3.37 (1H,
m, NH), 3.13 (2H, m, J ) 6.7, NHCH2), 2.36 (2H, t, J ) 7.2, CH2-
CO2H), 1.69 (2H, qn, J ) 7.1, NHCH2CH2), 1.60 (2H, qn, J ) 6.9,
CH2CH2CO2H), 1.37 (3H, t, J ) 7.2, CH2CH3).
Hapten C. The yield was 35%: 1H NMR (CDCl3) δ 7.66 (1H, s,
ar), 7.66 (1H, s, ar), 4.21 (2H, dxqxd, J ) 9.5, 7.2, and 2.1, CH2CH3),
3.27 (1H, m, NH), 3.11 (2H, dxq, J ) 11 and 6.9, NHCH2), 2.36 (2H,
t, J ) 7.3, CH2CO2H), 1.71-1.42 [6H, m, CH2(CH2)3CH2], 1.37 (3H,
txd, J ) 7.2 and 0.51, CH2CH3).
Hapten D. The yield was 69%: 1H NMR (CDCl3) δ 7.66 (1H, s,
ar), 7.55 (1H, d, J ) 1.1, ar), 4.21 (2H, m, J ) 7.2, CH2CH3), 3.34
(2H, m, NCH2), 2.87 (3H, d, J ) 8.4, NCH3), 2.43 (2H, t, J ) 7.2,
CH2CO2H), 1.92 (2H, qn, J ) 6.7, CH2CH2CH2), 1.35 (3H, t, J ) 7.1,
CH2CH3).
Figure 1. Structures of the haptens for bromophos-ethyl used for
immunization and antigen coating.
complete and incomplete adjuvants, and Sephadex G-25 were purchased
from Sigma (St. Louis, MO). Tetramethylbenzidine was obtained from
Boehringer Mannheim (Mannheim, Germany). Analytical (silica gel
F254) and preparative thin-layer chromatography (TLC) plates (silica
gel, 1 mm) were purchased from Merck (Darmstadt, Germany). The
dialysis membrane [molecular weight (MW) cutoff 12000-14000] was
obtained from Spectrum Laboratories (Rancho Dominguez, CA).
Microtiter plates (Maxisorp 442404) were purchased from Nunc
(Roskilde, Denmark). ELISA plates were washed with a model 1575
ImmunoWash, and well absorbances were read with a model 550 plate
reader, both from Bio-Rad (Hercules, CA). UV-vis spectra were
recorded on a Varian (Palo Alto, CA) Cary 3 spectrophotometer. NMR
spectra were obtained with a Bruker (Rheinstetten, Germany) ARX
spectrometer (300 MHz). Chemical shift values are given relative to
internal tetramethylsilane. Coupling constants are expressed in hertz,
and the abbreviations d, t, q, qn, sx, m, and ar represent doublet, triplet,
quartet, quintet, sextet, multiplet, and aromatic, respectively.
Synthesis of Haptens. The haptens used for immunization and
antigen coating are presented in Figure 1. The synthetic routes for the
haptens are illustrated in Figure 2. All of the haptens were purified by
column chromatography to give a single spot on TLC plates. The
procedure for the synthesis of hapten A was as follows.
III. The starting material 4-bromo-2,5-dichlorophenol (II) was
synthesized according to a published procedure (13). A solution of II
(0.92 g, 1.9 mmol) in 5 mL of acetonitrile was added dropwise to a
stirred mixture of 3.2 g (19.2 mmol) of ethyl dichlorothiophosphate
(I), 10 g of finely ground K2CO3, and 10 mL of acetonitrile. After 1 h
of stirring at room temperature (RT), the mixture was filtered through
Celite, and the solvent was removed under reduced pressure. The residue
was subjected to column chromatography [silica gel, hexane/benzene
(6:1)] to give 0.64 g (44%) of the product (III) as a colorless oil: 1H
NMR (CDCl3) δ 7.73 (1H, d, J ) 1.3, ar), 7.61 (1H, d, J ) 2.1, ar),
4.47 (2H, qxd, J ) 11.1 and 7.1, CH2CH3), 1.49 (3H, txd, J ) 7.1 and
1.0, CH2CH3).
Preparation of Hapten)Protein Conjugates. Haptens A and C
were covalently attached to BSA to be used as immunogens. Haptens
B and D were attached to OVA to be used as the coating antigens for
serum screening and competitive assay, respectively. Haptens A-D
were conjugated to horseradish peroxidase (HRP) to be used as enzyme
tracers. The method of conjugation used was the active ester method
(11). The procedure for the synthesis of this ester is described below.
Other active esters were synthesized by using the same procedure.
Hapten A (97 mg, 0.22 mmol) was dissolved in dichloromethane (5
mL) to which N-hydroxysuccinimide (28 mg, 0.24 mmol), N,N-
dicyclohexylcarbodiimide (63 mg, 0.24 mmol), and 4-(dimethylamino)-
pyridine (2.7 mg, 0.022 mmol) were added. The mixture was stirred
for 3 h and filtered, and the solvent was removed. Chromatography of
the resultant oil on silica gel using chlorform/ethyl acetate/acetic acid
(30:9:1) followed by preparative TLC using the same eluent gave the
active ester as a syrup (42 mg, 43%): 1H NMR (CDCl3) δ 7.67 (1H,
s, ar), 7.63 (1H, d, J ) 1.6, ar), 4.21 [2H, dxqxd, J ) 9, 7, and 1
(approximate values, peaks were not well resolved), CH2CH3], 3.56
(1H, m, NH), 3.25 (2H, dxq, J ) 13 and 6.8, NHCH2), 2.85 (4H, s,
succinyl), 2.73 (2H, t, J ) 7.2, CH2CO2H), 1.98 (2H, qn, J ) 7.0,
CH2CH2CH2), 1.37 (3H, t, J ) 7.1, CH2CH3).
1
Active Ester of Hapten B. Yield 39%; H NMR (CDCl3) δ 7.67
(1H, s, ar), 7.65 (1H, d, J ) 0.93, ar), 4.21 (2H, m, CH2CH3), 3.45
(1H, m, NH), 3.16 (2H, dxq, J ) 12 and 6.8, NHCH2), 2.85 (4H, s,
succinyl), 2.65 (2H, t, J ) 7.2, CH2CO2H), 1.82 (2H, qn, NHCH2CH2),
1.66 (2H, qn, J ) 7.1, CH2CH2CO2H), 1.37 (3H, t, J ) 7.1, CH2CH3).
1
Active Este of Hapten C. Yield 41%; H NMR (CDCl3) δ 7.67
(1H, s, ar), 7.65 (1H, d, J ) 0.93, ar), 4.21 (2H, dxqxd, J ) 4.7 and
2.2, CH2CH3), 3.57 (1H, m, NH), 3.26 (2H, dxq, J ) 13 and 6.8,
NHCH2), 2.85 (4H, s, succinyl), 2.73 (2H, t, J ) 7.2, CH2CO2H), 1.37
(3H, t, J ) 7.1, CH2CH3).
Hapten A. To a stirred solution of 100 mg (0.26 mmol) of III in 0.5
mL of methanol cooled in an ice-water bath was added dropwise a
solution of 38 mg (0.68 mmol) of KOH and 32 mg (0.31 mmol) of
4-aminobutyric acid in 0.25 mL of methanol. After stirring for 5 min,
the reaction mixture was filtered and extracted with 1 N HCl-
chloroform. The extract was dried over MgSO4, and the solvent was
evaporated. Column chromatography [silica gel, chloroform/ethyl
1
Active Ester of Hapten D. Yield 37%; H NMR(CDCl3) δ 7.66
(1H, d, J ) 0.8, ar), 7.55 (1H, d, J ) 1.5, ar), 4.16 (2H, dxqxd, J )
∼10, 7.1, and 2.4, CH2CH3), 3.39 (2H, m, NCH2), 2.89 (3H, d, J )
11, CH3N), 2.85 (4H, s, succinyl), 2.69 (2H, t, J ) 7.5, CH2CO2H),
2.04 (2H, qn, J ) 7.3, CH2CH2CH2), 1.36 (3H, txd, J ) 7.1 and 0.6,
CH2CH3).
Figure 2. Synthetic route for haptens.

ELISA for Bromophos-ethyl
J. Agric. Food Chem., Vol. 50, No. 23, 2002 6677
The procedures for coupling haptens to the carrier proteins were as
follows. To prepare hapten-BSA conjugates (immunogens), BSA (20
mg) was dissolved in 2 mL of borate buffer (0.2 M, pH 8.7) to which
0.4 mL of DMF was added. A solution of an active ester (16 mg, 0.03
mmol) dissolved in 0.1 mL of DMF was then added to the stirred protein
solution, and stirring was continued overnight at 4 °C. Hapten-OVA
conjugates (coating antigens) were prepared by using the same
procedure. Hapten-HRP conjugates (enzyme tracer) were prepared
according to the same procedure except that two hapten/protein molar
ratios (10 and 50) were employed. The conjugates were separated from
the uncoupled haptens by gel filtration (Sephadex G-25) using PBS
(10 mM phosphate buffer, 137 mM NaCl, 2.7 mM KCl, pH 7.4).
Finally, the eluates were dialyzed in water overnight and then freeze-
dried.
Immunization of Rabbits. Female New Zealand white rabbits were
immunized with hapten A- or hapten C-BSA. Routinely, 500 µg of
the conjugate dissolved in 500 µL of PBS was emulsified with Freund’s
complete adjuvant (1:1 volume ratio) and injected intradermally at
multiple sites on the back of each rabbit. After 2 weeks, each animal
was boosted with an additional 500 µg of the conjugate emulsified
with Freund’s incomplete adjuvant and bled 7-10 days later. After
this, boosting and bleeding were continued on a monthly basis. Serum
was separated from blood cells by centrifugation, and sodium azide
was added as a preservative at a final concentration of 0.02%. Serum
was then aliquotted and stored at -70 °C.
Competition curves were obtained by plotting absorbance against the
logarithm of analyte concentration. Sigmoidal curves were fitted to a
four-parameter logistic equation (14), from which IC50 values (con-
centration at which binding of the antibody to the coating antigen is
inhibited by 50%) were determined.
Competitive Direct Assay. Checkerboard assays, in which various
dilutions of sera were titrated against various amounts of enzyme tracers
(hapten A, C, or D conjugated to HRP), were used to select the most
suitable antiserum and enzyme tracer and to have a rough estimate of
their appropriate concentrations for competitive assays. The procedure
for the checkerboard assays was the same as that for competitive assays
(see below) except that only solvent instead of pesticide solution was
added at the competition step. After the most suitable antiserum and
enzyme tracer had been selected from the checkerboard assays, their
quantities for the competitive direct assays were optimized. The
tolerance of ELISA to various water-miscible organic solvents at the
competition step was tested by using the same procedure as that for
the indirect assay. The influence of phosphate ion concentration and
pH of assay buffer (20% methanol-PBS) on ELISA performance was
also studied using the same procedure as for the indirect assay. The
direct assays were performed as follows. All incubations except that
for precoating the wells with protein A were carried out at RT.
Microtiter plates were coated with protein A (5 µg/mL, 100 µL/well)
in carbonate-bicarbonate buffer (50 mM, pH 9.6) by overnight
incubation at 4 °C. The plates were washed five times with PBST and
were blocked by incubation with 100 µL/well of 1% gelatin in PBS
for 1 h. Then, the plates were washed and coated with 100 µL/well of
the antiserum dilutions in PBST for 1 h. After another washing step,
serial dilutions of the analyte in 10% MeOH-PBS were added (50
µL/well) followed by 50 µL/well of an enzyme tracer previously diluted
with PBS (77 ng/mL). After incubation for 1 h and another washing
step, 100 µL/well of a TMB solution was added. The reaction was
stopped after an appropriate time by adding 50 µL of 2 M H2SO4, and
absorbance was read at 450 nm. Competition curves were obtained by
using the same procedure as for the indirect assays.
Screening of Antisera. Several dilutions of each serum were titrated
against the coating antigen (hapten B-OVA, 1000 ng/well) to measure
the reactivity of antibodies. The procedure was similar to that for
checkerboard assays described below.
Competitive Indirect ELISA. Checkerboard assays, in which
several dilutions of sera were titrated against various amounts of the
coating antigen, were used to select the most suitable antiserum and to
have a rough estimate of appropriate antigen coating and antibody
concentrations for competitive assays. The checkerboard assays were
performed as follows. All incubations except that for antigen coating
were carried out at RT. Microtiter plates were coated with hapten
D-OVA (125-2000 µg/mL, 100 µL/well) in PBS (10 mM, pH 7.4)
by overnight incubation at 4 °C. The plates were washed five times
with PBST (10 mM PBS containing 0.05% Tween 20, pH 7.4) and
were blocked by incubation with 1% gelatin in PBS (200 µL/well) for
1 h. After another washing step, 100 µL/well of antiserum previously
diluted with PBST (1/1250-1/10000) was added. After incubation for
1 h, the plates were washed and 100 µL/well of a diluted (1/3000)
goat anti-rabbit IgG-HRP was added. The mixture was allowed to
incubate for 1 h, and after another washing step, 100 µL/well of a TMB
solution (400 µL of 0.6% TMB-DMSO and 100 µL of 1% H2O2
diluted with 25 mL of citrate-acetate buffer, pH 5.5) was added. The
reaction was stopped after 10 min by adding 50 µL of 2 M H2SO4, and
absorbance was read at 450 nm.
From the results of the checkerboard assays, an antiserum raised
against hapten A-BSA was selected as the most suitable one. Then,
the concentrations of the antibodies and the coating antigen (hapten
D-OVA) for competitive indirect assays were optimized. Additionally,
the tolerance of ELISA to various water-miscible organic solvents used
to dissolve pesticides was tested for assay optimization. For this test,
standard pesticide solutions were prepared in various concentrations
of acetone, acetonitrile, or methanol (10, 20, 40, and 80% in PBS, which
became 5, 10, 20, and 40%, respectively, after combination with equal
volumes of diluted antisera). The effect of buffering capacity of assay
solution on ELISA performance was also studied using different
concentrations of phosphate in 20% methanol-PBS to dissolve the
pesticide (10, 90, 190, and 390 mM phosphate, which became 10, 50,
100, and 200 mM, respectively, after combining with equal volumes
of the antisera diluted with 10 mM PBST). The influence of pH of the
assay solution, that is, 50 mM PBS with 20% methanol, was also
studied.
Determination of Cross-Reactivities. Several organophosphorus
pesticides and the metabolite of bromophos-ethyl (phenol) were tested
for cross-reactivity using both the indirect and direct ELISA procedures
described above. The cross-reactivity values were calculated as follows:
(IC50 of bromophos-ethyl/IC50 of compound) × 100
Analysis of Spiked Samples. Solutions of bromophos-ethyl in
methanol for the fortification of vegetable samples were prepared at
10, 50, 100, 500, and 1000 ng/mL. To 1 g of the finely chopped leaves
of the vegetables grown pesticide-free was added 1 mL of a fortification
solution. After being set aside for 24 h, the vegetable leaves were
incubated in 5 mL of methanol for 10 min with four vigorous shakes
and then filtered through Whatman no. 1 filter paper. The container
and the residues were rinsed with 5 mL of methanol and filtered, and
the filtrate was combined with the previous filtrate. Methanol was
evaporated to dryness under reduced pressure, and the residue was
resuspended in 10 mL of 20% methanol-PBS (50 or 10 mM for indirect
and direct assay, respectively). The extract was analyzed by both the
indirect and direct ELISAs.
Water samples from two different sources (tap water and pond water)
were spiked with bromophos-ethyl by adding 1 mL of a fortification
solution to 1 mL of membrane-filtered (0.25 µm) water samples. After
evaporation of the sample under a slow stream of nitrogen, the residue
was treated the same way as for vegetable samples.
RESULTS AND DISCUSSION
Hapten Selection and Synthesis. A suitable hapten for
immunization should preserve the structure of the target
compound as much as possible. The phosphorothioate organo-
phosphorus (OP) pesticides such as bromophos-ethyl have a
thiophosphate group in common and differ only in the structure
of the aromatic rings. Therefore, to achieve a high selectivity
in the bromophos-ethyl ELISA, it was desirable to synthesize
The procedure of the competition assay was as follows. To microtiter
plates coated and blocked as described above was added 50 µL/well
of serial dilutions of the analyte in methanol-PBS, followed by 50
µL/well of a previously determined antiserum dilution. After incubation
at RT for 1 h, antibody binding was assessed as described above.

6678 J. Agric. Food Chem., Vol. 50, No. 23, 2002
Kim et al.
Table 1. Influence of Organic Cosolvent, Phosphate Ion, and pH of
haptens having a bridge at the thiophosphate group preserving
the substitute groups on aromatic ring unique to bromophos-
ethyl. Haptens in this study have such structural features.
Heldman et al. (15) were the first to synthesize a hapten for an
OP pesticide with a spacer arm at the thiophosphate group.
However, a generic method was developed later by McAdam
et al. (16, 17). This method was applied to the synthesis of
haptens for the development of ELISAs of several OP pesticides
(7-12). This method requires a synthetic route involving seven
steps including protection and deprotection of both amino and
carboxyl groups. In an effort to simplify the synthetic process
for this class of haptens we developed a simpler generic method
that requires only two steps (7). It involves the reaction of
O-methyl (ethyl) dichlorothiophosphate with a phenol and K2-
CO3 in acetonitrile and the reaction of the substitution product
with an amino carboxylic acid (not protected) and KOH in
methanol (see Figure 2 for an example). Secondary as well as
primary amino acids could also be attached as the spacer arm.
This method was successfully applied to the synthesis of four
haptens for bromophos-ethyl (Figure 1). The reactions pro-
ceeded facilely with relatively high yields: 55 and 33-77% in
the first and second reactions, respectively. The reaction time
was relatively short: 1 h and a few minutes for the first and
second reactions, respectively. All of the haptens purified by
column chromatography were in a quite pure form, giving a
single spot on the TLC plate. This method was successfully
applied in this laboratory for the synthesis of haptens of several
other OP pesticides such as chlorpyrifos (7), chlorpyrifos-
methyl, diazinon, fenitrothion, parathion-methyl, and isofenphos
(18), and we applied for a patent. All of the carboxylic acid
haptens could be converted to the succinimide esters, active
esters for coupling haptens to carrier proteins.
a
the Assay Solution on Assay Parameters of Indirect ELISA
variable
acetone
concn
5%
10%
20%
40%
Absmax
slope
IC (ng/mL)
50
0.434
0.434
0.448
0.183
0.497
0.433
0.384
0.383
31
47
22
2.7
acetonitrile
methanol
phosphate ionsc
pH
5%
10%
20%
0.562
0.479
0.309
0.318
0.348
0.250
33
124
168
b
40%
5%
0.746
0.810
0.863
0.913
0.470
0.387
0.417
0.366
3.4
1.9
1.9
2.1
10%
20%
40%
10 mM
50 mM
100 mM
200 mM
0.835
0.823
0.670
0.645
0.389
0.360
0.390
0.374
9.1
7.1
16
14
6.0
6.5
7.0
7.4
8.0
8.5
0.667
0.714
0.841
0.861
0.838
0.694
0.253
0.246
0.299
0.331
0.266
0.275
12
28
15
12
17
21
a Assay conditions: antiserum to hapten A−BSA, diluted 1/1000 with 10 mM
PBST; coating antigen, hapten D−OVA, 150 ng/well; goat anti-rabbit IgG−HRP
diluted 1/2000. Data were obtained from the four-parameter sigmoidal fitting. Data
are the means of triplicates. b Data fitting was impossible due to poor color
development. c Final concentration of phosphate ions of the competition buffer
containing 138 mM NaCl and 2.7 mM KCl.
observed with acetone and acetonitrile. IC50 values in the
presence of acetone and acetonitrile were much higher than those
in the presence of methanol. Accordingly, we selected methanol
as the most suitable cosolvent. Several other workers reached
the same conclusion as ours in that methanol caused the least
negative effect on the performance of these assays. However,
their assays showed diverse direction and magnitude of response
to increasing concentration of methanol (19-22). Table 1 shows
that increasing the concentration of methanol causes an initial
increase followed by a decrease in sensitivity showing the lowest
IC50 value at 20% methanol. Table 1 also presents the effect
of the phosphate ion concentration at the competition step on
ELISA characteristics. Increasing the concentration of phosphate
ions caused an initial improvement followed by a decline in
assay sensitivity. The optimum concentration selected was 50
mM phosphate, showing the lowest IC50 value. Table 1 also
presents the effect of pH of assay solution on ELISA. The
physiological pH, pH 7.4, was selected as the optimum for the
assay.
Figure 3 shows a typical inhibition curve obtained under these
optimized conditions. The IC50 value of the assay was 3.9 ng/
mL with a detection limit of 0.3 ng/mL (20% inhibition). The
statistical detection limit estimated as the concentration that
correspond to the %B/B0 of the control (zero dose) minus 2
times the standard deviation of the control was 0.2 ng/mL. The
sensitivity of the indirect ELISA in terms of detection limit is
considerably higher than that of the GC method, which shows
a routine detection limit of 10 ng/mL for bromophos-ethyl (6).
Direct Competitive Assay. The checkerboard assays, in
which several dilutions of the sera were titrated against various
amounts of the enzyme tracers, were used to select the most
suitable antiserum and enzyme tracer. The final decision on
selecting the best combination was made from the results of
Competitive Indirect ELISA. The checkerboard assays, in
which several dilutions of the sera were titrated against various
amounts of the coating antigen (hapten D-OVA) were used to
select the most suitable antiserum and to optimize antigen
coating and antibody concentrations. Hapten D was used as a
coating hapten because it is heterologous to the immunizing
haptens in the structure of the bridge group. Hapten heterology
is commonly used to eliminate problems associated with the
strong affinity of the antibodies to the spacer arm that leads to
no or poor inhibition by the target compound. Heterology usually
results in weaker recognition of plate-coating antigens compared
with recognition of the target compound, allowing analyte at
low concentrations to compete with coating antigens. The
antiserum from hapten A was selected as the most suitable one
on the basis of its titer value which is the highest. The optimum
condition selected was the combination of the serum from hapten
A-BSA (third boost) diluted 1/1000 and the coating antigen
in 150 ng/well.
The use of organic solvents for extraction and/or solid phase
cleanup is very common in the analysis of nonpolar pesticide
residues in food and environmental samples, so it is desirable
to assess the effect of organic solvents on ELISA performance.
The effects of solvents (acetone, acetonitrile, and methanol) on
the ELISA system were evaluated by preparing standard curves
in buffers containing various amounts of organic solvent (5,
10, 20, and 40% in PBS). The results are presented in Table 1.
These solvents significantly influenced assay performance. The
speed of color development (estimated from Amax) in the
presence of acetone or acetonitrile was much slower compared
with that in the presence of methanol. It is interesting to note
that although the maximum absorbance was enhanced by
increasing the concentration of methanol, an opposite trend was

ELISA for Bromophos-ethyl
J. Agric. Food Chem., Vol. 50, No. 23, 2002 6679
Table 2. Influence of Organic Cosolvent, Phosphate Ion, and pH of
a
the Assay Solution on Assay Parameters of Direct ELISA
variable
acetone
concn
5%
10%
20%
40%
Absmax
slope
IC (ng/mL)
50
1.032
0.889
0.464
0.348
0.878
0.770
0.665
0.921
11
12
8.1
9.4
acetonitrile
methanol
phosphate ionsb
pH
5%
1.088
0.804
0.316
0.124
0.896
0.794
0.751
0.861
20
53
54
28
10%
20%
40%
5%
0.874
0.944
1.125
0.989
0.402
0.576
1.029
0.500
3.9
5.4
2.1
1.6
10%
20%
40%
10 mM
50 mM
100 mM
200 mM
0.964
0.835
0.831
0.885
0.981
0.928
0.974
0.885
11
28
25
16
6.0
6.5
7.0
7.4
8.0
8.5
0.685
0.798
0.881
0.880
0.873
0.954
1.118
1.114
1.286
1.173
1.123
1.157
32
20
23
17
24
23
Figure 3. ELISA competition curves of bromophos-ethyl by indirect
competitive assay. Reagent concentrations: antiserum (against hapten
A−BSA), 1/1000; coating antigen (hapten D−OVA), 150 ng/well; goat anti-
rabbit IgG−HRP, 1/3000. Each point represents the mean of 16
determinations. Vertical bars indicate ±SD about the mean.
a Assay conditions: precoating with protein A (0.5 µg/well); blocking with 1%
gelatin; antiserum to hapten A−BSA, diluted 1/2000 with 10 mM PBST; enzyme
tracer, hapten A−HRP, 3.8 ng/well. Data were obtained from the four-parameter
sigmoidal fitting. Data are the means of triplicates. b Final concentration of phosphate
ions of the competition buffer containing 138 mM NaCl and 2.7 mM KCl.
competitive assays for the various combinations, based on the
sensitivity of the assays (IC50 value and slope of the calibration
curve). The optimum combination selected was the antiserum
from hapten A-BSA (third boost) and the tracer hapten A-HRP
prepared at a 10:1 hapten/protein molar ratio. The antibody-
coated ELISA using this combination was optimized with regard
to the dilution of antiserum and enzyme tracer.
The effects of solvents (acetone, acetonitrile, and methanol)
on the ELISA system were evaluated by preparing standard
curves in buffers containing various amounts of solvent (5, 10,
20, and 40% in PBS). The results are presented in Table 2. As
observed in the indirect assay, the speed of color development
at the competition step decreased with increasing concentration
of acetone and acetonitrile, resulting in >50% retardation of
color development at 20 and 40% concentrations. Such an effect
was not observed with methanol. IC50 values in the presence of
acetone and acetonitrile were much higher than those in the
presence of methanol (Table 2). Therefore, methanol was the
most suitable cosolvent for the assay, in agreement with the
results of several other studies (23-25). Table 2 shows that
assay sensitivity continues to improve with increasing concen-
tration of methanol. However, 20% methanol was selected as
the optimum concentration, because the IC50 value at 20%
methanol (2.1 ng/mL) is close to that at 40% methanol (1.6
ng/mL) and the slope of the calibration curve is much sharper
at 20% methanol. Table 2 also presents the effect of the
concentration of the phosphate buffer in the competition solution
on ELISA characteristics. Increasing the concentration of the
phosphate caused initial improvement followed by a decline in
assay sensitivity. The optimum concentration selected was 10
mM phosphate, which showed the lowest IC50 value. Table 2
also presents the effect of pH of assay solution on ELISA. The
physiological pH, pH 7.4, was selected as the best one.
Figure 4. ELISA inhibition curves for bromophos-ethyl by direct competitive
assay. Reagent concentrations: precoating agent protein A, 0.5 µg/well;
antiserum (against hapten A−BSA), 1/2000; enzyme tracer (hapten
A−HRP), 3.8 ng/well. Each point represents the average of 16 determina-
tions. Vertical bars indicate ±SD about the mean.
the direct ELISA in terms of detection limit is considerably
higher than that of the GC method, which shows a routine
detection limit of 10 ng/mL for bromophos-ethyl (6).
Cross-Reactivity Studies. Several organophosphorus pesti-
cides as well as the metabolite of bromophos-ethyl (phenol)
were tested for cross-reactivities. Table 3 shows the cross-
reactivity that was found by both the indirect and direct assays,
expressed as a percentage of the IC50 of bromophos-ethyl. The
Figure 4 shows a typical inhibition curve obtained under the
optimized condition. The IC50 value of the assay was 6.5 ng/
mL with a detection limit of 1 ng/mL (20% inhibition). The
statistical detection limit (concentration that correspond to the
%B/B0 of the control minus 2 times the standard deviation) was
1.0 ng/mL. As observed in the indirect assay, the sensitivity of

6680 J. Agric. Food Chem., Vol. 50, No. 23, 2002
Kim et al.
Table 3. Cross-Reactivity of Compounds Structurally Related to Bromophos-ethyl Determined by Indirect and Direct Competitive ELISAsa
a Assay conditions were the same as those described in Table 1 (indirect ELISA) or 2 (direct ELISA). The standards were prepared in 20% MeOH−PBS (90 mM) or
10% MeOH−PBS (10 mM), respectively, and were combined with antisera diluted with 10 mM PBST. b IC50 values of the pesticides below chlorpyrifos-methyl could not be
determined accurately due to the limited solubility at high concentrations; however, it was clear that inhibition was <50% at 100 µg/mL. c Cross-reactivity (%) ) (IC50 of
bromophos-ethyl/IC50 of other compound) × 100.
interference with the assays was negligible except with bro-
mophos-methyl and chlorpyrifos in the direct assay. The
appreciable cross-reactivities of antibodies for these pesticides
are understandable, because they have the same or similar
aromatic structure as bromophos-ethyl. It is interesting to note
that when the same antiserum was used, there was significant
cross-reactivity to bromophos-methyl and chlorpyrifos when the
direct assay was used, whereas the cross-reactivity to these two
pesticides was much lower when the indirect assay was used.
The result can be rationalized by assuming that recognition of
the antibodies to the aromatic ring structure of the antigen
relative to the thiophosphate structure is higher in the direct
assay, which would allow bromophos-methyl and chlorpyrifos
to compete with the competitor (enzyme tracer) more effectively
in the direct assay. It may be concluded that the competitive
ELISAs that were developed are suitable for the sensitive and
selective detection of bromophos-ethyl, with the exception of
bromophos-methyl and chlorpyrifos in the direct assay.
(sesame/indirect and kale/indirect were exceptions). The range
of recovery for all combinations was between 82 and 128%.
The plot of the recovered pesticide against the spiked pesticide
for all combinations gave regression lines with slopes and
intercepts very close to 1 and 0 ng/mL, respectively. Fortification
of vegetable samples was carried out by incubating the chopped
vegetable leaves in fortification solutions for 24 h. Thus,
pesticide may have penetrated quite deeply into the interior of
the leaves during the incubation period. However, the recovery
data suggest that pesticide can be recovered to near perfection
by using the extraction procedure adopted in this study.
Recoveries from tap water and pond water determined by both
indirect and direct ELISAs are presented also in Table 4. The
range of recovery was between 95 and 127%. The plot of the
recovered pesticide against the spiked pesticide gave regression
lines with slopes and intercepts very close to 1 and 0 ng/mL,
respectively. Despite the simple pretreatment taken for sample
preparation (evaporation of liquid and dissolution of the residue
in buffer), no significant matrix effect was observed. Overall,
the ELISAs developed in this study can accurately determine
bromophos-ethyl residues in vegetable and water samples after
the simple and rapid extraction procedure used in this study.
Comparison of the Indirect and Direct Assays. The
sensitivity of the indirect assay in terms of IC50 value is a bit
Recovery Studies. Three kinds of vegetables and waters from
two different sources were spiked with bromophos-ethyl and
analyzed by both indirect and direct ELISAs. Recoveries of the
pesticide from spiked vegetable samples are presented in Table
4. Of six combinations of the kind of vegetables and assay
format, four showed recoveries ranging from 89 to 112%

ELISA for Bromophos-ethyl
J. Agric. Food Chem., Vol. 50, No. 23, 2002 6681
EnVironmental Analysis; Van Emon, J., Mumma, R., Eds.; ACS
Symposium Series 442; American Chemical Society: Washing-
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Table 4. Recovery of Bromophos-ethyl Spiked into Vegetable and
Water Samplesa
recoveries by
fortified concn
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sample
lettuce
(ng/mL)
indirect ELISA
direct ELISA
10
50
100
500
1000
101
104
91
99
102
107
105
108
98
99
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kale
10
50
100
500
1000
113
100
128
117
103
89
111
107
109
98
sesame
pond water
tap water
10
50
100
500
1000
97
101
96
98
82
100
99
108
92
111
10
50
100
500
1000
99
99
97
96
96
102
101
106
107
115
10
50
100
500
1000
102
103
110
112
112
99
127
100
95
104
a Assay conditions were the same as those described in Table 3. Data are the
means of triplicates. The slopes of the least-squares plot of recovered versus spiked
concentration (log−log), in respective order, were 0.999, 0.980, 0.995, 1.022, 0.972,
1.007, 0.959, 1.147, 1.123 and 1.026.
better than that of the direct assay (IC50 values of 3.9 vs 6.5
ng/mL). However, the direct assay gives much sharper standard
curves (slopes of 0.4 vs 0.9). As far as selectivity is concerned,
the indirect assay is better, that is, lower cross-reactivity of the
antibodies. Therefore, if sensitivity is more important than
selectivity in an analysis, then the direct assay should be chosen.
If samples are suspected of containing multiple organophos-
phorus pesticides, then the direct assay would be the better one
to use.
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Absmax, maximum absorbance; BSA, bovine serum albumin;
DMF, dimethylformamide; DMSO, dimethyl sulfoxide; ELISA,
enzyme-linked immunosorbent assay; IC50, concentration of
analyte giving 50% inhibition of the maximum absorbance; IgG,
immunoglobulin G; NMR, nuclear magnetic resonance; OVA,
ovalbumin; PBS, phosphate-buffered saline; PBST, phosphate-
buffered saline-0.05% Tween 20; TLC, thin-layer chromatog-
raphy; TMB, 3,3′,5,5′-tetramethylbenzidine.
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Received for review May 29, 2002. Revised manuscript received August
25, 2002. Accepted August 29, 2002. This work was supported by Korea
Research Foundation Grant KRF-2000-GA0023. Additional support
from U.S. NIEHS Superfund Basic Research Program P42 ES04699
and U.S. NIEHS Environmental Health Center P30 ES05707 is also
acknowledged.
JF025703+