Hapten and antibody production for a sensitive immunoassay determining a human urinary metabolite of the pyrethroid insecticide permethrin

Article abstract of DOI:10.1021/jf049646r

Permethrin is the most popular synthetic pyrethroid insecticide in agriculture and public health. For the development of the enzyme-linked immunosorbent assay (ELISA) to evaluate human exposure to permethrin, the glycine conjugate (DCCA-glycine) of a major metabolite, cis/trans-3-(2,2- dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (DCCA), of permethrin was established as the target analyte. Four different types of the cis- and trans-isomers of immunizing haptens were synthesized as follows: N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine (hapten 3), N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1- carbonyl)-4-amino-L-phenyl-alanine (hapten 5), N-(N-(cis/trans-3-(2,2- dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine)-amino-6-(2, 4-dinitrophenyl)aminohexanoic acid (hapten 9), and N-(cis/trans-3-(2,2- dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine-4-oxobutanoic acid (hapten 24). Sixteen polyclonal antibodies produced against each cis- or trans-hapten-thyroglobulin conjugate as immunogens were screened against numerous hapten-bovine serum albumin conjugates as coating antigens. Six ELISAs with both a heterologous hapten structure and a heterologous hapten configuration (cis/trans or trans/cis) between antibody and coating antigen showed a high sensitivity for the target analyte. The IC₅₀ was 1.3, 2.1, and 2.2 μg/L for the trans-target analyte and 0.4, 2.3, and 2.8 μg/L for the cis-target analyte. The immunizing haptens, except for hapten 5, provided the target specific antibodies. Molecular modeling of the haptens supported the selection of reasonable immunizing haptens that best mimicked the target analyte. Hapten 5 was suitable as a coating antigen rather than as an immunogen since it had a different geometry. Very low cross-reactivities were measured to permethrin, its free metabolite (DCCA), PBA-glycine conjugate, and glycine. The ELISA will be optimized for the detection of total cis/trans-DCCA-glycine in human urine samples.

Full text of DOI:10.1021/jf049646r

J. Agric. Food Chem. 2004, 52, 4583−4594 4583
Hapten and Antibody Production for a Sensitive Immunoassay
Determining a Human Urinary Metabolite of the Pyrethroid
Insecticide Permethrin
KI CHANG AHN, TAKAHO WATANABE, SHIRLEY J. GEE, AND
BRUCE D. HAMMOCK*
Department of Entomology and UCD Cancer Research Center, University of California,
Davis, California 95616
Permethrin is the most popular synthetic pyrethroid insecticide in agriculture and public health. For
the development of the enzyme-linked immunosorbent assay (ELISA) to evaluate human exposure
to permethrin, the glycine conjugate (DCCA-glycine) of a major metabolite, cis/trans-3-(2,2-
dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (DCCA), of permethrin was established as
the target analyte. Four different types of the cis- and trans-isomers of immunizing haptens were
synthesized as follows: N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine
(hapten 3), N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)-4-amino-L-phenyl-
alanine (hapten 5), N-(N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)glycine)-
amino-6-(2,4-dinitrophenyl)aminohexanoic acid (hapten 9), and N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-
dimethylcyclopropane-1-carbonyl)glycine-4-oxobutanoic acid (hapten 24). Sixteen polyclonal antibodies
produced against each cis- or trans-hapten-thyroglobulin conjugate as immunogens were screened
against numerous hapten-bovine serum albumin conjugates as coating antigens. Six ELISAs with
both a heterologous hapten structure and a heterologous hapten configuration (cis/trans or trans/cis)
between antibody and coating antigen showed a high sensitivity for the target analyte. The IC50 was
1.3, 2.1, and 2.2 µg/L for the trans-target analyte and 0.4, 2.3, and 2.8 µg/L for the cis-target analyte.
The immunizing haptens, except for hapten 5, provided the target specific antibodies. Molecular
modeling of the haptens supported the selection of reasonable immunizing haptens that best mimicked
the target analyte. Hapten 5 was suitable as a coating antigen rather than as an immunogen since
it had a different geometry. Very low cross-reactivities were measured to permethrin, its free metabolite
(DCCA), PBA-glycine conjugate, and glycine. The ELISA will be optimized for the detection of total
cis/trans-DCCA-glycine in human urine samples.
KEYWORDS: ELISA; permethrin; glycine conjugate; human exposure; hapten
INTRODUCTION
Environmental Protection Agency has classified permethrin as
a potential carcinogen (12). It also belongs to categories of
endocrine disrupting compounds (13), priority pollutants (14,
15), and environmental contaminants.
The pyrethroid insecticide permethrin acts as a neurotoxin
to control a wide range of insects in agriculture, forestry, homes,
horticulture, and public health around the world (1-5). Per-
methrin is very nontoxic to mammals, whereas it is highly toxic
to some nontarget insects such as honeybees and other beneficial
insects (6). This compound has apparent toxic effects to some
aquatic species such as fish, aquatic insects, crayfish, and shrimp
at parts per billion levels (7-9). Its high octanol-water partition
coefficient suggests that it may have a tendency to bioaccumu-
late in living organisms (10). In the study on the neonatal effect
of the exposure, Cantalamessa (11) suggested that permethrin
is more acutely toxic to children than to adults. Because
permethrin causes lung and liver tumors in mice, the U.S.
Permethrin is the most popular insecticide among the pyre-
throid insecticides in the United States as the active ingredient
in personal care products, such as shampoos and lotions for
lice. Permethrin residues have been found in ground and surface
waters throughout the United States. It is also detected in
agricultural products, particularly spinach, tomatoes, celery,
lettuce, and peaches, and peach and plum baby food (16). The
most common route of exposure to pyrethroid insecticides for
the general population is through drinking water and the
ingestion of foods such as vegetables and fruits onto which the
insecticide has been applied. Farmers, pesticide applicators,
manufacturers, and military service personnel may also receive
additional occupational exposure through dermal contact and
* To whom correspondence should be addressed. Tel: 530-752-7519.
Fax: 530-752-1537. E-mail: bdhammock@ucdavis.edu.
10.1021/jf049646r CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/25/2004

4584 J. Agric. Food Chem., Vol. 52, No. 15, 2004
Ahn et al.
inhalation. For humans, pyrethroid insecticides are much less
toxic than many other insecticides. The ADI values (acceptable
daily intake for man) range from 0.01 (deltamethrin) to 0.05
(permethrin and cypermethrin) mg/kg body weight per day (17,
18). However, overexposure causes reversible symptoms such
as headache, dizziness, nausea, irritation of the skin and nose,
and paraesthesia (19, 20).
In mammals, permethrin, like other pyrethroid insecticides,
is rapidly metabolized by ester cleavage to its constituent acids
(cis- and trans-DCCA) and alcohol (PBOH) and subsequent
oxidation of the alcohol to PBA. There is little tendency for it
to accumulate in tissues. These metabolites might be partially
conjugated to glycine and/or glucuronic acid and then finally
excreted in the urine (21-23), which is a common pathway for
xenobiotics.
In humans, the major metabolic pathway is ester hydrolysis
followed by conjugation. Ring hydroxylation, oxidation at the
gem-dimethyl group, and other oxidation pathways result in
minor metabolites (24). The major permethrin metabolites, cis-
and trans-DCCA, and PBA were determined to serve as
biomarkers in the urine of exposed people (25). While little is
known about the conjugates of pyrethroid metabolites in
humans, it has been well-established that glycine is the most
common amino acid used in conjugation reactions with free
carboxylic acids of xenobiotics (26). The glucuronide and
glycine conjugates of DCCA in rat and mouse and the
glucuronide, glycine, serine, glutamine, and glutamic acid
conjugates of DCCA in cockroach, housefly, and cabbage looper
have been found (27).
Analytical methods for detection of pyrethroid metabolites
in biological samples such as urine and blood include some
sample preparation steps such as acid hydrolysis, liquid-liquid
or solid phase extraction, and derivatization with high-
performance liquid chromatography or gas chromatography with
mass spectrometry (25, 28-30).
Although the instrumental methods are very sensitive for these
metabolites, they are time-consuming and expensive and not
suitable for a routine and rapid analysis. Immunoassay tech-
niques are widely used in clinical diagnostics, environmental
monitoring, food quality, agriculture, and field or on-site test
of personnel. An example of the latter is deployed soldiers
exposed to toxic chemicals. The assays are rapid, sensitive, and
selective analytical tools to determine trace chemicals such as
agrochemicals and their metabolites as key urinary biomarkers
of exposure (27).
A sensitive ELISA with an IC50 value of 0.4 µg/L and a
detection range of 0.04-10 µg/L in buffer was developed for
the glycine conjugate of the pyrethroid metabolite PBA (PBA-
glycine) (32). The limit of quantitation was 1 µg/L in real human
urine.
It is likely that the relative amount of DCCA-glycine
conjugate is higher than that of the PBA-glycine, because the
PBOH that is directly hydrolyzed from the parent can enter
several metabolic pathways including direct conversion to the
glucuronide conjugate prior to further oxidation to PBA.
Therefore, DCCA-glycine can exist in high abundance and then
be a biomarker as an indicator of exposure to this insecticide
and a good target analyte for biomonitoring in human urine.
The preparation of haptens as immunogens and coating
antigens is one of the major stages in the development of
immunoassays for small molecules (31, 33). The synthesis of
haptens can sometimes require extensive investment of time
even before Ab production. Therefore, the hapten chemistry
should be thoroughly considered in order to develop sensitive
and selective immunoassays. In this study, the authors report
the syntheses of cis/trans-haptens and their use for Ab produc-
tion and ELISA development. Because the target analyte
possesses separate cis- and trans-isomers of DCCA-glycine,
optimal haptens would produce antibodies specific to each
isomer of the target analyte as well as for use as coating antigens.
Various haptens with different cis- or trans-configurations were
synthesized and used to produce polyclonal antibodies in rabbits.
The screening of the antibodies against the various coating
antigens and characterization of the antibodies are reported.
MATERIALS AND METHODS
Chemicals. The cis- and trans-DCCA, as the starting materials for
hapten syntheses, were prepared, according to published synthetic
procedures (34-36). Organic materials for the synthesis were purchased
from Aldrich Chemical Co. (Milwaukee, WI) and Fisher Scientific
(Pittsburgh, PA). Thin-layer chromatography (TLC) utilized 0.2 mm
precoated silica gel 60 F254 on glass plates from E. Merck (Darmstadt,
Germany), and detection was by ultraviolet light or iodine vapor stain.
Column chromatographic separations were carried out using Baker silica
gel (40 µm average particle size) using the indicated solvents where
the f notation denotes a stepwise concentration gradient. The coupling
reagents were purchased from Aldrich. BSA, Thyr, GAR-HRP as the
second Ab, Tween 20, and 3,3′,5,5′-TMB were purchased from Sigma
Chemical Co. (St. Louis, MO).
Instruments. 1H nuclear magnetic resonance (NMR) spectra of
compounds synthesized were obtained on a General Electric QE-300
spectrometer (Bruker NMR, Billerica, ME) using tetramethylsilane as
an internal standard. Electrospray mass spectra in the positive (MS-
ESI+) or negative (MS-ESI-) mode were recorded by a Micromass
Quattro Ultima triple quadrupole tandem mass spectrometer (Micro-
mass, Manchester, United Kingdom). Melting points were deter-
mined on a Thomas-Hoover Uni-Melt apparatus (Thomas Scientific,
Swedesboro, NJ) and are uncorrected. Rf values refer to TLC on silica
gel 60 F254, precoated plates (Merck) with visualization under exposure
to either ultraviolet light (254 nm) or iodine vapor. ELISA was
performed on 96 well microtiter plates (Nunc-Immuno plate, MaxiSorp
surface, Roskilde, Denmark) and read spectrophotometrically with a
microplate reader (Molecular Devices, Menlo Park, CA) in the dual
wavelength mode (450-650 nm).
Hapten Synthesis and Verification. The proposed target haptens
are shown in Figure 1. Because the target DCCA-glycine is of a small
molecular weight (MW 265) and requires conjugation to carrier proteins
in order to be immunogenic, various haptens with the carboxylic group
or amine group were synthesized. The reactions followed the procedures
used in previous publications (32, 37).
N-(cis/trans-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane-1-
carbonyl)glycine (cis/trans-DCCA-glycine, cis/trans-Hapten 3; Fig-
ure 2). Thionyl chloride (SOCl2, 3 mL) was added to cis-DCCA (1500
mg, 7.17 mmol) in a 10 mL round bottomed flask. The mixture was
stirred under N2 at 65 °C for 1.5 h. The solution was concentrated
under reduced pressure to remove excess SOCl2. Hexane was added,
and then the solution was concentrated again. The crude acid chloride,
cis-2, was obtained as a pale yellow liquid. It was added dropwise to
a vigorously stirred ice-cooled solution of glycine in 10 mL of 2 N
KOH. A 1 N solution of KOH was added to keep the mixture slightly
basic. After 3 h, the odor of the acid chloride was no longer detectable.
The mixture was acidified with 6 N HCl and extracted twice with ethyl
acetate (30 mL). The combined organic phase was dried over anhydrous
sodium sulfate, and the solvent was removed under reduced pressure.
The residue was recrystallized from the mixture of ethyl acetate and
tert-butyl chloride to give 1084 mg (56%) of cis-DCCA-glycine, cis-
3, as a white solid; mp 141-143 °C. TLC [ethyl acetate/hexane/acetic
1
acid (1:1:0.1, v/v/v)] Rf, 0.85. H NMR (DMSO-d6): δ 1.15 (s, 1H,
CH3), 1.32 (s, 3H, CH3), 1.85 (d, J ) 8.5 Hz, 1H, CHCO), 2.03 (dd,
J ) 8.7, 8.7 Hz, CdC-CH), 3.78 (t, J ) 5.8 Hz, 2H, NCH2), 6.06 (d,
J ) 8.9 Hz, 1H, CdCH), 8.40 (t, J ) 5.8 Hz, 1H, NH). MS-ESI- m/z
calcd for [M - H]- ) C10H13Cl2NO3, 264.03; observed, 264.09.
The trans-isomer of compound 3 (1023 mg, 53%) was prepared as
a white solid from the trans-DCCA by the same method as described

ELISA for a Human Urinary Metabolite of Permethrin
J. Agric. Food Chem., Vol. 52, No. 15, 2004 4585
Figure 1. Target analyte and haptens designed.
Figure 2. Scheme for the synthesis of the target analyte (cis/trans-DCCA-glycine, hapten 3) and cis/trans-hapten 5.
above for the cis-isomer of compound 3; mp 178-179 °C. TLC (ethyl
acetate/hexane/acetic acid [1:1:0.1, v/v/v)] Rf, 0.75. 1H NMR (DMSO-
d6): δ 1.14 (s, 3H, CH3), 1.15 (s, 3H, CH3), 1.83 (d, J ) 5.3 Hz, 1H,
CHCO), 2.04 (dd, J ) 5.3 Hz, 8.2 Hz, 1H, CdC-CH), 3.78 (t, 5.6
Hz, 2H, NCH2), 6.04 (d, J ) 8.3 Hz, 1H, CdCH), 8.36 (t, J ) 5.8 Hz,
1H, NH). MS-ESI- m/z calcd for [M - H]- ) C10H13Cl2NO3, 264.03;
observed, 263.95.
cis/trans-Compound 4 (Figure 2). Crude acid chloride, cis-2 (0.06
mmol), was added dropwise to a vigorously stirred ice-cooled solution
of 4-nitro-L-phenylalanine (139 mg, 0.66 mmol) in 10 mL of 2 N KOH.

4586 J. Agric. Food Chem., Vol. 52, No. 15, 2004
Ahn et al.
Figure 3. Scheme for the synthesis of cis/trans-hapten 9.
Over the course of 30 min, 1 N KOH was added to keep the mixture
slightly basic. After 3 h, the odor of the acid chloride was no longer
detectable. The mixture was acidified with 6 N HCl and extracted twice
with ethyl acetate (30 mL). The combined organic phase was dried
over anhydrous sodium sulfate, and the solvent was removed under
reduced pressure. The residue was purified by using preparative TLC
on silica gel with the mixture of ethyl acetate/hexane/acetic acid (1:
1:0.01, v/v/v) as an eluent to give 180 mg of cis-compound 4 as a
yellow oil. TLC [ethyl acetate/hexane/acetic acid (1:1:0.1, v/v/v)] Rf,
0.19. 1H NMR (CDCl3): δ 1.22 (s, 3H, CH3), 1.33 (s, 3H, CH3), 1.63
(d, J ) 8.5 Hz, 1H, CHCO), 2.27 (dd, J ) 8.7, 8.8 Hz, 1H, CdC-
CH), 3.22-3.26 (m, 2H, NCHCH2Ar), 4.95 (dt, J ) 7.1, 5.8 Hz, 1H,
NCHCO), 5.63 (d, J ) 8.9 Hz, 1H, CdCH), 7.26-7.40 (m, 4H, Ar),
sulfate. The solvent was removed under reduced pressure. The oil
residue was purified by using preparative TLC on silica gel with the
mixture of methanol/methylene chloride/acetic acid (1:9:0.2, v/v/v) as
an eluent to give 112 mg (47%) of cis-hapten 5 as a yellow solid;
mp 93-97 °C. TLC [methanol/methylene chloride/acetic acid (1:9:
1
0.2, v/v/v)] Rf, 0.59. H NMR (DMSO-d6): δ 1.15 (s, 3H, CH3), 1.32
(s, 3H, CH3), 1.86 (d, J ) 8.6 Hz, 1H, CHCO), 2.03 (dd, J ) 8.7,
8.7 Hz, 1H, CdC-CH), 3.32-3.48 (m, 2H, NCHCH2Ar), 4.28 (dt,
J ) 7.0, 5.6 Hz, 1H, NCHCO), 6.05 (d, J ) 8.8 Hz, 1H, CdCH),
6.99-8.13 (m, 4H, Ar), 8.40 (d, J ) 7.1 Hz, 1H, NH). MS-ESI- m/z
calcd for [M - H]- ) C17H20Cl2N2O3, 369.09; observed, 369.10.
By the same method as described above for the cis-isomer of hapten
5, 98 mg (41%) of trans-hapten 5 was prepared as a yellow solid from
the trans-compound 4; mp 102-105 °C. TLC [methanol/methylene
chloride/acetic acid (1:9:0.2, v/v/v)] Rf, 0.59. 1H NMR (DMSO-d6): δ
1.20 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.75 (d, J ) 5.0 Hz, 1H, CHCO),
2.11 (dd, J ) 5.5, 8.4 Hz, 1H, CdC-CH), 3.34-3.50 (m, 2H,
NCHCH2Ar), 4.52 (dt, J ) 7.3, 5.5 Hz, 1H, NCHCO), 6.07 (d, J )
8.5 Hz, CdCH), 6.99-8.13 (m, 4H, Ar), 8.23 (d, J ) 7.2 Hz, 1H,
NH). MS-ESI- m/z calcd for [M - H]- ) C17H20Cl2N2O3, 369.09;
observed, 369.04.
8.20 (d, J ) 7.1 Hz, 1H, NH). MS-ESI- m/z calcd for [M - H]-
)
C17H18Cl2N2O5, 399.06; observed, 399.24.
The trans-isomer of compound 4 (150 mg) was prepared as a yellow
oil from the trans-acid chloride by the same method as described above
for the cis-isomer of compound 4. TLC [ethyl acetate/hexane/acetic
acid (1:1:0.1, v/v/v)] Rf, 0.20. 1H NMR (CDCl3): δ 1.23 (s, 3H, CH3),
1.35 (s, 3H, CH3), 1.63 (d, J ) 5.4 Hz, 1H, CHCO), 2.26 (dd, J ) 5.4,
8.4 Hz, 1H, CdC-CH), 3.21-3.25 (m, 2H, NCHCH2Ar), 4.91 (dt,
J ) 7.3, 5.5 Hz, 1H, NCHCO), 5.63 (d, J ) 8.4 Hz, 1H, CdCH),
7.28-7.42 (m, 4H, Ar), 8.23 (d, J ) 7.2 Hz, 1H, NH). MS-ESI- m/z
calcd for [M - H]- ) C17H18Cl2N2O5, 399.06; observed, 399.24.
N-(cis/trans-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane-1-
carbonyl)-4-amino-L-phenylalanine (cis/trans-Hapten 5; Figure 2).
The mixture of cis-compound 4 (261 mg, 0.65 mmol) and stannous
chloride dihydrate (733 mg, 3.25 mmol) in 2 mL of ethanol was stirred
at 70 °C in an oil bath for 30 min. The mixture was cooled and poured
into the slurry of water, ethyl acetate, and Celite. NaHCO3 was added
in portions. The neutral mixture was filtered, and the solids on the
filter and in the flask were washed with water and ethyl acetate. The
ethyl acetate phase was separated and dried over anhydrous sodium
cis/trans-Compound 8 (Figure 3). Compound 7 as a yellow solid
was synthesized according to Watanabe et al. (37). TLC [methanol/
methylene chloride (1:9, v/v)] Rf, 0.63.
In a 25 mL flask, cis-DCCA-glycine (79.84 mg, 0.3 mmol),
1-hydroxy-1H-benzotriazole monohydrate (81.08 mg, 0.2 mmol), and
compound 7 (97.89 mg, 0.3 mmol) were dissolved in 2 mL of dry
tetrahydrofuran (THF), and then, 41.8 µL of triethylamine (0.3 mmol)
and N,N′-dicyclohexylcarbodiimide (68.09 mg, 0.33 mmol) dissolved
in 500 µL of dry THF were added to the mixture. The reaction mixture
was stirred for 1 h in an ice bath and for 3 h at room temperature and
then was filtered to remove the dicyclohexylurea. Ethyl acetate (50
mL) was added and was washed with saturated sodium bicarbonate

ELISA for a Human Urinary Metabolite of Permethrin
J. Agric. Food Chem., Vol. 52, No. 15, 2004 4587
Figure 4. Scheme for the synthesis of cis/trans-haptens 24 and 26.
1
solution, saturated ammonium chloride solution, saturated sodium
bicarbonate solution, and water (50 mL of each). After the organic
phase was dried over anhydrous sodium sulfate, ethyl acetate was
removed under reduced pressure. The resultant residue was recrystal-
lized from ether to give 140 mg of cis-compound 8 as a yellow solid.
methylene chloride (1:9, v/v)] Rf, 0.59. H NMR (CDCl3): δ 1.25 (s,
3H, CH3), 1.27 (s, 3H, CH3), 1.50-1.91 (m, 8H, NHCH2CH2CH2 and
CHCO), 2.23 (dd, J ) 5.3 Hz, 8.2 Hz, 1H, CdC-CH), 4.01 (t, 5.7
Hz, 2H, NCH2CO), 4.03-4.16 (m, 1H, CHCO), 4.61 (q, J ) 14.5 Hz,
2H, CH2CH), 5.61 (d, J ) 8.3 Hz, 1H, CdCH), 6.83 (t, J ) 5.7 Hz,
1H, CONHCH2), 6.93-8.55 (m, 3H, Ar). MS-ESI- m/z calcd for
[M - H]- ) C22H27Cl2N5O8, 558.12; observed, 558.35.
cis/trans-Compound 23 (Figure 4). The mixture of the cis-DCCA-
glycine, cis-3 (150 mg, 0.56 mmol), 4-bromo-butyric acid tert-butyl
ester (249.88 mg, 1.12 mmol), and potassium carbonate (111.63 mg,
0.616 mmol) in 1 mL of anhydrous DMF was reacted at 100 °C for 3
h. The resulting mixture was filtered to remove excess K2CO3 and HBr
produced in the reaction. The filtrate diluted with 20 mL of ethyl acetate
was washed twice with 20 mL of distilled water. The organic layer
was dried over anhydrous sodium sulfate, and the solvent was removed
by evaporation. The residue was chromatographed on silica gel eluting
with the mixture of ethyl acetate/hexane (1:2, v/v). Fractions containing
pure product by TLC were stripped under high vacuum to obtain 179
mg of cis-compound 23 as a transparent oil. TLC [ethyl acetate/hexane
1
TLC [methanol/methylene chloride (0.5:9.5, v/v)] Rf, 0.60. H NMR
(CDCl3): δ 1.21 (s, 3H, CH3), 1.27 (s, 3H, CH3), 1.51-1.96 (m, 8H,
NHCH2CH2CH2 and CHCO), 2.27 (dd, J ) 8.6 Hz, 8.7 Hz, 1H, CdC-
CH), 3.80 (s, 3H, OCH3), 4.01 (t, 5.8 Hz, 2H, NCH2CO), 4.08-4.18
(m, 1H, CHCO), 4.65 (q, J ) 14.7 Hz, 2H, CH2CH), 5.64 (d, J ) 8.9
Hz, 1H, CdCH), 6.82 (t, J ) 5.8 Hz, 1H, CONHCH2), 6.92-8.58 (m,
3H, Ar). MS-ESI+ m/z calcd for [M + H]+ ) C23H29Cl2N5O8, 574.14;
observed, 574.12.
By the same method as described above for the cis-isomer of
compound 8, 158 mg of the trans-isomer of compound 8 was prepared
as a yellow solid from the trans-DCCA-glycine, trans-3. TLC
[methanol/methylene chloride (0.5:9.5, v/v)] Rf, 0.60. 1H NMR
(CDCl3): δ 1.21 (s, 3H, CH3), 1.24 (s, 3H, CH3), 1.51-1.94 (m, 8H,
NHCH2CH2CH2 and CHCO), 2.25 (dd, J ) 5.3 Hz, 8.2 Hz, 1H, CdC-
CH), 3.78 (s, 3H, OCH3), 4.05 (t, 5.7 Hz, 2H, NCH2CO), 4.08-4.18
(m, 1H, CHCO), 4.64 (q, J ) 14.5 Hz, 2H, CH2CH), 5.63 (d, J ) 8.3
Hz, 1H, CdCH), 6.80 (t, J ) 5.7 Hz, 1H, CONHCH2), 6.91-8.57 (m,
3H, Ar). MS-ESI+ m/z calcd for [M + H]+ ) C23H29Cl2N5O8, 574.14;
observed, 574.12.
1
(1:2, v/v)] Rf, 0.67. H NMR (CDCl3): δ 1.25 (s, 3H, CH3), 1.35 (s,
3H, CH3), 1.45 (s, 9H, 3CH3), 1.50 (d, J ) 8.5 Hz, 1H, CHCO), 2.03
(dd, J ) 8.7, 8.7 Hz, 1H, CdC-CH), 1.90-2.05 (m, 2H, CH2CH2CH2),
2.31 (t, 2H, CH2CH2CH2), 4.03 (t, 2H, CH2CH2CH2), 4.20 (d, J ) 5.7
Hz, 2H, NCH2COO), 6.01 (bs, 1H, NH), 6.41 (d, J ) 8.8 Hz, 1H,
CdCH).
N-(N-(cis/trans-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane-
1-carbonyl)glycine)amino-6-(2,4-dinitrophenyl)aminohexanoic Acid
(cis/trans-Hapten 9; Figure 3). The hydrolysis reaction of cis-
compound 8 (100 mg, 0.17 mmol) was conducted in 10 mL of 1,4-
dioxane/water (1:1, v/v) with 10 equiv of lithium hydroxide mono-
hydrate. After 4.5 h, the resulting mixture was acidified to pH 4 with
6 N HCl and extracted twice with ethyl acetate (30 mL). The combined
organic phase was washed with distilled water (30 mL) and dried over
anhydrous sodium sulfate. The solvent was removed under reduced
pressure. The residue was purified by using preparative TLC on silica
gel with the mixture of methanol/methylene chloride (1:9, v/v) as an
eluent to give 80 mg (84%) of cis-hapten-9 as a yellow solid; mp 50-
55 °C. TLC [methanol/methylene chloride (1:9, v/v)] Rf, 0.59. 1H NMR
(CDCl3): δ 1.18 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.53-1.95 (m, 8H,
NHCH2CH2CH2 and CHCO), 2.25 (dd, J ) 8.6 Hz, 8.7 Hz, 1H, CdC-
CH), 3.97 (t, 5.8 Hz, 2H, NCH2CO), 4.08-4.15 (m, 1H, CHCO), 4.63
(q, J ) 14.7 Hz, 2H, CH2CH), 5.63 (d, J ) 8.9 Hz, 1H, CdCH), 6.78
(t, J ) 5.8 Hz, 1H, CONHCH2), 6.90-8.55 (m, 3H, Ar). MS-ESI-
m/z calcd for [M - H]- ) C22H27Cl2N5O8, 558.12; observed, 558.29.
By the same method as described above for the cis-isomer of hapten
9, 75 mg (79%) of trans-hapten 9 was prepared as a yellow solid from
the trans-isomer of compound 8; mp 65-70 °C. TLC [methanol/
By the same method as described above for the cis-isomer of
compound 23, the trans-isomer of compound 23 (179 mg) was prepared
as a transparent oil from the trans-isomer of DCCA-glycine, trans-3.
1
TLC [ethyl acetate/hexane (1:2, v/v)] Rf, 0.64. H NMR (CDCl3): δ
1.20 (s, 3H, CH3), 1.33 (s, 3H, CH3), 1.44 (s, 9H, 3CH3), 1.61 (d, J )
5.4 Hz, 1H, CHCO), 2.05 (dd, J ) 5.4, 8.7 Hz, 1H, CdC-CH), 1.92-
2.07 (m, 2H, CH2CH2CH2), 2.35 (t, 2H, CH2CH2CH2), 4.10 (t, 2H,
CH2CH2CH2), 4.18 (d, J ) 5.7 Hz, 2H, NCH2COO), 6.10 (bs, 1H,
NH), 6.42 (d, J ) 8.3 Hz, 1H, CdCH).
N-(cis/trans-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane-1-
carbonyl)glycine-4-oxobutanoic Acid (cis/trans-Hapten 24; Figure
4). Trifluoroacetic acid (TFA) (0.5 mL) was added to the cis-ester 23,
and the mixture was allowed to stand at ambient temperature for 15
min. The TFA was stripped off under vacuum, and ethyl acetate was
added twice and stripped to remove residual TFA. The residue was
chromatographed on silica gel eluting with the mixture of ethyl acetate/
hexane (1:1, v/v). Fractions containing pure product by TLC were
stripped under high vacuum, and the residue was recrystallized from
ethyl acetate and tert-butyl chloride to give 37 mg (19%) of cis-hapten-
24 as a white solid; mp 85-88 °C. TLC [ethyl acetate/hexane/acetic
acid (1:1:0.1, v/v/v)] Rf, 0.55. 1H NMR (CDCl3): δ 1.25 (s, 3H, CH3),

4588 J. Agric. Food Chem., Vol. 52, No. 15, 2004
Ahn et al.
1.27 (s, 3H, CH3), 1.71 (d, J ) 8.5 Hz, 1H, CHCO), 1.96-2.02 (m,
3H, CdC-CH and CH2CH2CH2), 2.40 (t, 2H, CH2CH2CH2), 3.99
(t, 2H, CH2CH2CH2), 4.23 (d, J ) 5.7 Hz, 2H, NCH2COO), 4.72
(bs, 1H, NH), 6.38 (d, J ) 8.8 Hz, 1H, CdCH). MS-ESI+ m/z calcd
for [M + H]+ ) C14H18Cl2NO5, 352.06; observed, 352.09.
By the same method as described above for the cis-isomer of hapten
24, 150 mg (76%) of trans-hapten 24 from the trans-isomer of ester
23 was prepared as a white solid; mp 103-104 °C. TLC [ethyl acetate/
hexane/acetic acid (1:1:0.1, v/v/v)] Rf, 0.55. 1H NMR (CDCl3): δ 1.25
(s, 3H, CH3), 1.27 (s, 3H, CH3), 1.52 (d, J ) 5.4 Hz, 1H, CHCO),
2.09-2.17 (m, 3H, CdC-CH and CH2CH2CH2), 2.57 (t, 2H,
CH2CH2CH2), 4.12 (t, 2H, CH2CH2CH2), 4.30 (d, J ) 5.7 Hz, 2H,
NCH2COO), 5.70 (d, J ) 8.8 Hz, 1H, CdCH), 6.21 (bs, 1H, NH).
MS-ESI+ m/z calcd for [M + H]+ ) C14H18Cl2NO5, 352.06; observed,
352.19.
in 4 drops of ethanol and treated with 1 mL of 1 N HCl. The resulting
solution was stirred in an ice bath as 0.5 mL of 0.20 M sodium nitrite
was added. DMF (0.4 mL) was then added dropwise to give a
homogeneous solution, which was divided into two equal aliquots.
Twenty-five milligrams of Thyr or BSA was dissolved in 5.5 mL of
0.05 M borate buffer (pH 9.6) and 1.0 mL of DMF. Aliquots of the
activated hapten solution were added dropwise to the two stirred protein
solutions. The reaction mixture was stirred at 4 °C overnight, and the
resulting yellow conjugates were purified by exhaustive dialysis in
normal strength phosphate-buffered saline (1 × PBS), which was
changed for a new buffer twice a day for 4 days. Finally, the conjugates
were dispensed into the 2 mL cryogenic vials and stored at -80 °C.
NHS Method for Conjugation of Haptens with Carboxylic Acid to
Proteins. cis/trans-DCCA-glycine and cis/trans-haptens 9, 24, and 26
(0.04 mmol) were dissolved in 1 mL of dry DMF with sulfo-NHS (0.06
mmol) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydro-
chloride (0.05 mmol). After the mixture was stirred at room temperature
overnight, the activated hapten was added slowly to the protein solution
(25 mg of protein in 6 mL of 0.05 M borate buffer at pH 8) with
vigorous stirring. The reaction mixture was purified as described above.
Immunization and Antiserum Preparation. The immunization was
made by following the protocol reported previously (40, 41). Two
female New Zealand white rabbits were immunized for each isomer
of immunogen (rabbits 3700 and 3701 for cis-hapten 3-Thyr, rabbits
3702 and 3703 for trans-hapten 3-Thyr, rabbits 3704 and 3705 for
cis-hapten 5-Thyr, rabbits 3706 and 3707 for trans-hapten 5-Thyr,
rabbits 3708 and 3709 for cis-hapten 9-Thyr, rabbits 3710 and 3711
for trans-hapten 9-Thyr, rabbits 369 and 3696 for cis-hapten 24-
Thyr, and rabbits 3697 and 3698 for trans-hapten 24-Thyr). Each
immunogen (100 µg) in 0.5 mL of 0.85% saline was emulsified with
an equal volume of Freund’s complete adjuvant, and then, the emulsion
was injected subcutaneously. After 3 weeks, the animals were boosted
with an additional 100 µg of immunogen that was emulsified with
Freund’s incomplete adjuvant (1:1, v/v). The boosts were given every
3 weeks, and blood samples were drawn 7 days after each boost to
check the titer of antibodies. The final serum was collected 5 months
following the first immunization. The blood was collected into the
Vacutainer tube with a serum separation gel. The antiserum was
obtained by centrifugation and stored at -80 °C. The antiserum was
used without any purification.
ELISA. Buffer Solutions. Normal strength PBS [1 × PBS; 8 g/L of
sodium chloride (NaCl), 0.2 g/L of potassium phosphate, monobasic
(KH2PO4), 1.2 g/L of sodium phosphate dibasic anhydrous (Na2HPO4),
and 0.2 g/L of potassium chloride (KCl), pH 7.5], PBST [1 × PBS
containing 0.05% (v/v) Tween 20, pH 7.5], 0.1 × PBST (PBS diluted
1:10 with distilled water and containing 0.05% Tween 20), carbonate
buffer [1.59 g/L sodium carbonate (Na2CO3), 2.93 g/L sodium hydrogen
carbonate (NaHCO3), pH 9.6], 0.05 M borate buffer (19.1 g/L Na2B4O7‚
10H2O, pH 8), and 0.05 M acetate buffer (14.71 g/L Na3C6H5O7‚2H2O)
were used for immunoassay.
ELISA. Indirect ELISA and competitive indirect ELISA were
performed according to the method of Voller et al. (42). The 96 well
microtiter plates were coated overnight at 4 °C with 100 µL/well of
the appropriate coating antigen concentration in 0.1 M carbonate-
bicarbonate buffer (pH 9.6). After it was washed five times with PBST,
the plate was incubated with 200 µL/well of a 0.5% BSA solution in
PBS for 1 h at room temperature. After another washing step, 100 µL/
well of antiserum diluted in PBST per well (for titration experiment)
or 50 µL/well of antiserum diluted in PBST and 50 µL/well of standard
analyte and sample solution were added, mixed for 30 s in the reader,
and incubated for 1 h at room temperature. The standard analyte
concentrations ranged from 0.003 µg/L to 5 mg/L. After the plate was
washed, 100 µL/well of the secondary Ab GAR-HRP (1:5000 in PBST)
was added and incubated for 1 h at room temperature. The plate was
washed, and 100 µL/well of a substrate solution (0.1 mL of 1%
hydrogen peroxide and 0.4 mL of 0.6% 3,3′,5,5′-TMB in 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/well of 2 N sulfuric acid. The absorbance was measured using
a dual wavelength mode at 450 minus 650 nm. Standard curves were
obtained by plotting absorbance against the logarithm of analyte
cis/trans-Compound 25 (Figure 4). The mixture of cis-DCCA-
glycine, cis-3 (100 mg, 0.38 mmol), tert-butyl bromoacetate (148.25
mg, 0.76 mmol), and potassium carbonate (76.11 mg, 0.418 mmol) in
1 mL of anhydrous DMF was reacted at 100 °C for 3 h. The resulting
mixture was further prepared by the same method as described above
for hapten 23. The residue was chromatographed on silica gel eluting
with the ethyl acetate/hexane (1:3, v/v) mixture. The fractions containing
pure product identified by TLC were stripped under high vacuum. After
toluene was added, the solvent was removed under reduced pressure
to give 126 mg of cis-compound 25 as a white solid. TLC [ethyl acetate/
1
hexane (1:2, v/v)] Rf, 0.64. H NMR (CDCl3): δ 1.24 (s, 3H, CH3),
1.27 (s, 3H, CH3), 1.48 (s, 9H, 3CH3), 1.61 (d, J ) 8.5 Hz, 1H, CHCO),
2.02 (dd, J ) 8.7, 8.7 Hz, 1H, CdC-CH), 4.16 (d, J ) 5.7 Hz, 2H,
NCH2COO), 4.58 (s, 2H, COOCH2COO), 6.07 (bs, 1H, NH), 6.40 (d,
J ) 8.7 Hz, 1H, CdCH).
By the same method as described above for the cis-isomer of
compound 25, 138 mg of the trans-isomer of compound 25 was
prepared as a white solid from the trans-DCCA-glycine. TLC [ethyl
1
acetate/hexane (1:2, v/v)] Rf, 0.53. H NMR (CDCl3): δ 1.20 (s, 3H,
CH3), 1.23 (s, 3H, CH3), 1.50 (s, 9H, 3CH3), 1.53 (d, J ) 5.4 Hz, 1H,
CHCO), 2.03 (dd, J ) 5.3, 8.3 Hz, 1H, CdC-CH), 4.12 (d, J ) 5.7
Hz, 2H, NCH2COO), 4.70 (s, 2H, COOCH2COO), 6.23 (bs, 1H, NH),
6.41 (d, J ) 8.7 Hz, 1H, CdCH).
N-(cis/trans-3-(2,2-Dichlorovinyl)-2,2-dimethylcyclopropane-1-
carbonyl)glycine-oxoacetic Acid (cis/trans-Hapten 26; Figure 4).
TFA (0.5 mL) was added to the cis-ester 25, and the mixture was
allowed to stand at ambient temperature for 15 min. By the same method
as described above for hapten 24, the mixture was further prepared,
chromatographed on silica gel eluting with the mixture of ethyl acetate/
hexane (1:3 f 1:1, v/v), and recrystallized to give 65 mg (53%) of
cis-hapten 26 as a white solid; mp 120-123 °C. TLC [ethyl acetate/
hexane/acetic acid (1:1:0.1, v/v/v)] Rf, 0.38. 1H NMR (CDCl3): δ 1.20
(s, 3H, CH3), 1.27 (s, 3H, CH3), 1.61 (d, J ) 8.5 Hz, 1H, CHCO),
2.04 (dd, J ) 8.7, 8.7 Hz, 1H, CdC-CH), 4.18 (d, J ) 5.7 Hz,
2H, NCH2COO), 4.58 (s, 2H, COOCH2COO), 6.17 (bs, 1H, NH), 6.42
(d, J ) 8.7 Hz, 1H, CdCH). MS-ESI+ m/z calcd for [M + H]+
C12H15Cl2NO5, 324.03; observed, 324.03.
)
By the same method as described above for cis-hapten 26, 75 mg
(61%) of the trans-isomer of hapten 26 was prepared as a white solid
from the trans-compound 25; mp 123-125 °C. TLC [ethyl acetate/
hexane/acetic acid (1:1:0.1, v/v/v)] Rf, 0.15. 1H NMR (CDCl3): δ 1.24
(s, 3H, CH3), 1.27 (s, 3H, CH3), 1.43 (d, J ) 5.4 Hz, 1H, CHCO),
2.13 (dd, J ) 5.3, 8.3 Hz, 1H, CdC-CH), 4.13 (d, J ) 5.7 Hz, 2H,
NCH2COO), 4.77 (s, 2H, COOCH2COO), 6.25 (bs, 1H, NH), 6.38
(d, J ) 8.7 Hz, 1H, CdCH). MS-ESI+ m/z calcd for [M + H]+
C12H15Cl2NO5, 324.03; observed, 324.03.
)
Hapten Conjugation. Hapten-protein conjugates were synthesized
using the water soluble carbodiimide method (38) for haptens with a
carboxylic acid and the diazotization method (39) for haptens with an
amine group. For immunogens, cis/trans-DCCA-glycine (cis/trans-
hapten 3), cis/trans-hapten 5, cis/trans-hapten 9, and cis/trans-hapten
24 were conjugated to Thyr. For coating antigens, cis/trans-DCCA and
cis/trans-hapten 26 as well as the above haptens were conjugated to
BSA.
Diazotization Method for Conjugation of cis/trans-Hapten 5 with
Amine Group to Proteins. cis/trans-Hapten 5 (0.10 mmol) was dissolved

ELISA for a Human Urinary Metabolite of Permethrin
J. Agric. Food Chem., Vol. 52, No. 15, 2004 4589
concentration, which were fitted to a four parameter logistic equation:
y ) {(A - D)/[1 + (x/C)B]} + D where A is the maximum absorbance
at no analyte, B is the curve slope at the inflection point, C is the IC50,
and D is the minimum absorbance at infinite concentration. The IC50
value was expressed as the sensitivity of the immunoassay.
Molecular Modeling. Molecular modeling was done using the CS
Chemoffice 6.0 software package (CambridgeSoft Corporation, Cam-
bridge, MA). The geometries optimized of the cis-target analyte and
cis-haptens at their minimum energy levels with the minimum RMS
gradient of 0.1 were calculated by semiempirical quantum mechanics
MNDO model (43) with a wave function of close shell (restricted).
The best immunizing and coating haptens on the target analyte are
evaluated with both real data (Table 2) and modeling.
Cross-Reactivity (CR). The CR studies were evaluated by using
the standard solution of the permethrin metabolites and other structurally
related compounds. The test compounds are listed in Table 3. The CR
was calculated as (IC50 of the target analyte/IC50 of the tested compound)
× 100.
RESULTS AND DISCUSSION
Hapten Design and Synthesis. Possessing two chiral centers
in the cyclopropane ring of the permethrin molecule, permethrin
has a mixture of four different optical and geometrical isomers.
It is mainly classified into two pairs of isomers with different
geometries, referred to as the cis- and the trans-isomers. The
target analyte, cis/trans-DCCA-glycine, in itself cannot be used
as an immunogen because of its low molecular weight (MW
265.1). Therefore, haptens mimicking the target analyte and
containing reactive groups such as a -COOH or -NH2 group
for conjugation to carrier proteins were synthesized to develop
an immunoassay. One of the strategies for designing the
immunizing hapten was to link the spacer through the -COOH
group of the target molecule and leave the two Cl atoms and
the amide bond (-RCONH-) and carbonyl group (-RCO-)
in the molecule to improve Ab specificity. The target analyte
(DCCA-glycine) molecule also possesses a cyclopropane ring
that is also divided into the cis- and trans-isomers such as the
parent permethrin. As another strategy, the haptens were
designed so that each individual cis- and trans-isomer was
obtained. This is both for the production of Ab specific to each
isomer of the target and for the enhancement of sensitivity with
heterologous cis/trans or trans/cis configurational immunoassay
format that for example uses the cis-isomer for coating antigen
with Ab raised against the trans-isomer of the immunogen.
For the production of a specific Ab capable of recognizing
N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-
carbonyl)glycine, four different types of haptens as immunizing
antigens were prepared. The target analyte (cis/trans-DCCA-
glycine) with the carboxylic acid also served as cis/trans-hapten
3 with no spacer. It was synthesized via the Schotten-Boumann
reaction with a synthetic procedure similar to that used for the
PBA-glycine conjugate (32) (Figure 2). That is, the acid chloride
of DCCA was reacted with glycine in alkaline solution and then
acidified to obtain cis/trans-DCCA-glycine. N-(cis/trans-3-(2,2-
dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)-4-amino-
L-phenylalanine (hapten 5) was synthesized to preserve the
carboxylic acid group of the target acid. Briefly, the acid chloride
of cis/trans-DCCA was also reacted via the Schotten-Boumann
reaction with 4-nitro-L-phenylalanine to give the N-acylated nitro
intermediate 4. Subsequent reduction of the nitro group with
stannous chloride gave hapten 5. To get high titer of Ab more
rapidly (37), N-(N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-dimeth-
ylcyclopropane-1-carbonyl)glycine)amino-6-(2,4-dinitrophenyl)-
aminohexanoic acid (hapten 9) was synthesized by coupling Nꢀ-
2,4-dinitrophenyl-L-lysine to the target analyte according to the
Figure 5. Optimized molecular model of (A,E) DCCA-glycine, (B,F) hapten
5, (C,G) hapten 9, and (D,H) hapten 24; A−D, space filling models, and
E−H, stick models.
synthetic method by Watanabe et al. (37) (Figure 3). Finally,
as a common modification of the acid moiety on the target,
cis/trans-DCCA-glycine was reacted with bromo-butyric acid
tert-butyl ester to give ester intermediate 23. Subsequent alkaline
hydrolysis with lithium hydroxide and acidification gave the
four carbon spacer linker of N-(cis/trans-3-(2,2-dichlorovinyl)-
2,2-dimethylcyclopropane-1-carbonyl)glycine-4-oxobutanoic acid
(hapten 24) (Figure 4).

4590 J. Agric. Food Chem., Vol. 52, No. 15, 2004
Ahn et al.
Table 1. Antiserum Titer Response of Rabbits against Various Coating Antigensa
coating antigen
trans- cis-
cis-
trans-
cis-
trans-
cis-
trans-
cis-
trans-
cis-
trans-
immunogen
antiserum 1−BSA 1−BSA 3−BSA 3−BSA 5−BSA 5−BSA 9−BSA 9−BSA 24−BSA 24−BSA 26−BSA 26−BSA
cis-hapten 3−Thyr
3700
3701
3702
3703
3704
3705
3706
3707
3708
3709
3710
3711
3696
369
+++
+++
+++
+++
++
+++
+++
+++
+
++
−
+
+++
+++
++
+++
+++
+++
+++
++
+++
+++
+++
++
+++
++
++
+++
+++
+++
+++
+++b
+++b
+++
+++
++
+++
+++
+++
+++
+++
+
+++
+++
+++b
+++b
++
+++
+++
+++
+++
+++
+
+++
+++
+++
+++
+++
+++
+++
++
+++
+++b
+++b
+++
+++
−
+
−
−
+++
+++
−
+++
++
+++
+++
+++
+++
++
+++
+++
+++
+++b
+++b
+++
+++
+++
+++
++
+++
+++
+++
+++
++
+++
+++
+++
+++
+++
+++b
+++b
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
++
+++
+++
+++
+++
+++
+++
+++
+++
+
++
+++
++
+++
+++
+++b
+++b
+++
+++
+++
+++
+++
+++
+++
+++
+
+++
+
+
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
trans-hapten 3−Thyr
cis-hapten 5−Thyr
trans-hapten 5−Thyr
cis-hapten 9−Thyr
trans-hapten 9−Thyr
cis-hapten 24−Thyr
trans-hapten 24−Thyr
+++
+++
+++
+++
+++b
+++b
−
−
−
+
++
+
+++
+++
+++
+++
+++
+++
+
+++b
+++b
+++
+++
3698
3699
+++
−
++
+++
a The data shown are at a coating antigen concentration of 1 µg/mL and an Ab dilution of 1:32 000; −, absorbance < 0.3; +, absorbance 0.3−0.6; ++, absorbance
0.6−0.9; and +++, absorbance > 0.9. b Homologous systems.
Additionally cis/trans-hapten 26 with a carboxylic acid was
synthesized according to the same method as hapten 24. cis/
trans-DCCA-glycine was reacted with bromo-acetic acid tert-
butyl ester to give ester intermediate 25. The alkaline hydrolysis
and acidification gave N-(cis/trans-3-(2,2-dichlorovinyl)-2,2-
dimethylcyclopropane-1-carbonyl)glycine-2-oxoacetic acid (hap-
ten 26) with a shorter two carbon space linker to serve as a
coating antigen.
Molecular Modeling of Haptens. Although the planar
chemical structures of haptens are shown to completely mimic
the target analyte, their geometries can be different. The
geometry study of the molecules is useful to select an immuniz-
ing hapten to produce Ab against the target analyte (44-46).
The immunizing haptens synthesized were characterized by
computing the molecules at their minimum energy levels.
Observing the molecular models (Figure 5), the geometries of
hapten 24 and the target analyte were completely matched, and
the geometry of hapten 9 showed a small difference in the
cyclopropane ring moiety of the molecules of the target and
hapten 9. On the contrary, that of hapten 5 did not exhibit any
similar geometry to the model of the target analyte.
Hapten Conjugation. For immunogens, haptens with a
carboxylic acid group were conjugated to Thyr by the sulfo-
NHS ester method and with an amino group by the diazotization
method. The sulfo-NHS conjugation method was used to obtain
the more stable sulfo-NHS ester intermediate as compared to
an NHS ester and thus more increased coupling yield (47). In
this study, the carrier protein Thyr was chosen as an immunizing
protein (48). For coating antigens, haptens were conjugated to
BSA by the NHS ester and diazotization methods. This avoids
recognition of protein-urea adducts that form with the use of
water soluble diimides. trans-Hapten 9-protein conjugates were
confirmed by the UV/vis spectrophotometry. trans-Hapten 9
showed a characteristic peak at 360 nm distinguishing it from
that of each protein. The hapten-protein conjugates also showed
a peak at 360 nm. These results indicate that hapten was
successfully conjugated to the protein by the sulfo-NHS method.
In addition, the yellow color of the hapten 5- and 9-protein
conjugates was an indicator of a successful coupling reaction,
whereas haptens 1, 3, and 24 as white solids did not show any
characteristic peak distinguished from that of protein. From the
available amino groups according to the TNBS method of
Habeeb (49), 20-61% of the cis/trans-haptens 1, 3, and 24 were
conjugated in the hapten-Thyr or -BSA conjugates, suggesting
successful hapten loading on the proteins.
Titers and Screening of the Antisera. For the production
of Ab specific to the cis/trans-target analyte, the cis/trans-
hapten-Thyr conjugates were injected a total of seven times
into each rabbit. The antisera collected after each boost were
subjected to titration by the homologous indirect ELISA. All
of the antisera showed relatively constant high titers after the
fifth immunization (data not shown). These results indicate that
specific antibodies in the rabbit antiserum were produced against
each immunogen. The final serum was collected 5 months
following the first immunization and was used for the subse-
quent screening in search of antibodies specific to the target
compound. Despite showing high titers of antisera after the fifth
immunization, rabbits were boosted twice more to raise more
sensitive and specific antibodies. None of the antisera demon-
strated any significant affinity for BSA itself as a coating
antigen.
The checkerboard titration was used for the screening of Ab
and antigen combinations. All antisera had high titers in
homologous systems, but in the heterologous systems, antisera
produced against hapten 9-Thyr and trans-hapten 24-Thyr
conjugates showed especially low titers (absorbance < 0.3)
against the coating antigens, hapten 5-BSA and cis-hapten
5-BSA, respectively (Table 1).
All of the 16 antisera were screened against 12 coating
antigens via the simple inhibition test at the two concentrations
(10 and 1000 µg/L) of each individual cis- or trans-target
analyte. There was no or low inhibition in homologous systems,
whereas there were much higher inhibitions in heterologous
systems (data not shown). The combinations of Ab and coating
antigen with over 80% inhibition at the concentration of 1000
µg/L of the target analyte and with over 20% inhibition at the
concentration of 10 µg/L were again screened at the 10 different
concentrations of each target analyte.
As shown in Table 2, none of the antibodies generated against
hapten 5-Thyr had IC50 values below 3 µg/L for either the
trans- or the cis-target analyte. The antisera specific to the target
trans-DCCA-glycine were mainly raised against trans-haptens
3-, 9-, and 24-Thyr except for trans-hapten 5-Thyr. This
result suggests that because the spacer arm of hapten 5 possessed
a bulky aminophenyl functional group that may serve as an
antigenic determinant, the hapten 5-Thyr conjugate appeared

ELISA for a Human Urinary Metabolite of Permethrin
J. Agric. Food Chem., Vol. 52, No. 15, 2004 4591
Table 2. Combination Data for Screening with Competitive Indirect
ELISA against cis- or trans-DCCA-glycinea
combination
IC
50
analyte
immunogen
antiserum/coating antigen (µg/L)
trans-DCCA-glycine trans-hapten 3−Thyr Ab 3702/cis-hapten 5−BSA
Ab 3703/cis-hapten 5−BSA
26
1.2
42
trans-hapten 5−Thyr Ab 3706/cis-hapten 9−BSA
Ab 3706/cis-hapten 26−BSA 50
Ab 3707/cis-hapten 1−BSA
Ab 3707/cis-hapten 9−BSA
52
7.6
Ab 3707/cis-hapten 26−BSA 16
trans-hapten 9−Thyr Ab 3710/cis-hapten 5−BSA
Ab 3710/trans-hapten 5−BSA
1.2
5.8
81
Ab 3711/cis-hapten 1−BSA
Ab 3711/trans-hapten 5−BSA 25
trans-hapten 24−Thyr Ab 3698/cis-hapten 3−BSA
Ab 3698/cis-hapten 5−BSA
93
2.0
Ab 3698/trans-hapten 5−BSA 14
Ab 3699/cis-hapten 1−BSA
Ab 3699/cis-hapten 5−BSA
Ab 3699/trans-hapten 5−BSA
Ab 3699/cis-hapten 9−BSA
Ab 3700/cis-hapten 5−BSA
Ab 3700/trans-hapten 5−BSA
Ab 3701/cis-hapten 5−BSA
Ab 3701/trans-hapten 5−BSA
Ab 3704/cis-hapten 1−BSA
Ab 3709/cis-haptne 1−BSA
Ab 3709/trans-hapten 1−BSA
15
2.0
4.1
8.7
18
5.8
39
3.5
84
10
3.5
cis-DCCA-glycine cis-hapten 3−Thyr
cis-hapten 5−Thyr
cis-hapten 9−Thyr
Ab 3709/trans-hapten 3−BSA 92
Ab 3709/cis-hapten 5−BSA
Ab 3709/trans-hapten 5−BSA
2.9
0.7
Ab 3709/trans-hapten 26−BSA 23
cis-hapten 24−Thyr Ab 369/cis-hapten 5−BSA 35
Ab 369/trans-hapten 5−BSA 3.7
a The analytes were prepared in the PBS solution.
to produce spacer specific antibodies, which resulted in lower
specificity to the target analyte as compared to other Ab groups.
Showing low IC50 values (<2 µg/L) for the trans-target analyte,
the Ab coating antigen combinations of Ab 3703/cis-hapten
5-BSA, Ab 3710/cis-hapten 5-BSA, and Ab 3698/cis-hapten
5-BSA were chosen for more study. The antisera specific to
cis-DCCA-glycine were also generated against the cis-haptens
3-, 9-, and 24-Thyr. Showing low IC50 values (<3 µg/L)
for the cis-target analyte, the combinations of Ab 3701/trans-
hapten 5-BSA, Ab 3709/trans-hapten 5-BSA, and Ab 369/
trans-hapten 5-BSA were also chosen. These results suggest
that the structure of hapten 5 was not suitable to produce the
Ab specific to the target but was suitable as a coating antigen
in a heterologous system. With respect to the isomeric config-
uration of the molecule, the heterologous cis/trans or trans/cis
configuration systems between Ab and coating antigen were
much more sensitive than homologous configuration systems.
This result was consistent with studies of immunoassays for
the pyrethroids permethrin and cypermethrin (36, 50). These
assays also used the heterologous configuration. For an example,
the combination of the Ab raised against the trans-permethrin
hapten was used with a cis-coating antigen for a sensitive
immunoassay.
Figure 6. Six ELISA inhibition curves for trans-DCCA-glycine (A) and
cis-DCCA-glycine (B) under unoptimized conditions. The reagent con-
centrations were as follows: the assay for trans-DCCA-glycine using Ab
3703, 1:32 000; Ab 3710, 1:8000; and Ab 3698, 1:16 000; and coating
antigen cis-hapten 5−BSA, 1, 5, and 2 µg/mL, respectively. The assay
for cis-DCCA-glycine using Ab 3701, 1:16 000; Ab 3709, 1:8000; and Ab
369, 1:16 000; and coating antigen trans-hapten 5−BSA, 1 µg/mL. The
dilution ratios of the Ab are the final concentrations in the wells.
affinity for the trans-configuration of target analyte than the
cis-configuration, whereas the antibodies generated against the
cis-configuration had a higher affinity for the cis-target analyte.
The CRs of the tested trans-antibodies to cis-DCCA-glycine
were below 28%. The Ab 3703 showed relatively higher CR to
cis-DCCA-glycine than that of other testes antibodies, Ab 3710
and Ab 3698, indicating the mixture of cis/trans-DCCA-glycine
can be analyzed using this Ab. Additionally, the shorter linker
of hapten presents the hapten relatively close to the protein
carrier surface; thus, the Ab produced against the hapten can
provide broader selectivity to the structurally related compounds
(47). This theory was applicable to Ab 3703. Antibodies
produced against the trans-isomer of haptens 9 and 24 with
longer linkers showed a lower CR to the cis-target analyte. The
Ab (Ab 3703) raised against trans-hapten 3 (trans-DCCA-
glycine) with no spacer displayed a better recognition to the
cis-isomer of the target analyte than that of other antibodies
Inhibition curves of the target analyte with six combinations
of the antiserum and the coating antigen (Figure 6) resulted in
IC50 values of 1.3, 2.1, and 2.2 µg/L for trans-DCCA-glycine
and 0.4, 2.3, and 2.8 µg/L for cis-DCCA-glycine under
unoptimized conditions, indicating that the sensitive immuno-
assay for the analysis of the target analyte can be developed.
CR. As seen in Table 3, the antibodies generated against
the immunizing haptens of the trans-configuration had a higher

4592 J. Agric. Food Chem., Vol. 52, No. 15, 2004
Ahn et al.
hapten structure and configuration systems between antibodies
and coating antigen. The IC50 values are as low as 1.3-2.2 µg/L
for trans-DCCA-glycine and 0.4-2.8 µg/L for cis-DCCA-
glycine. Depending in part on the application, the optimum Ab
for the assay will be selected. The Ab 3703 generated against
the trans-immunogen provided broad selectivity for the cis-target
analyte. The ELISA with the combination of Ab 3703 and
coating antigen, cis-hapten 5-BSA, can be good for detection
of the cis- and trans-isomers of DCCA-glycine in the sample.
The immunoassay will be optimized with the parameters such
as detergent, solvent content, pH, and ionic strength in the assay
buffer for the best sensitivity and reproducibility, providing a
powerful tool for human monitoring for pyrethroid exposure.
Table 3. CR (%) of the Selected Antibodies to Permethrin Metabolites
and Other Structurally Related Compoundsa
IC (CR [%])
50
Ab 3703/cis-
5−BSA
Ab 3710/cis-
5−BSA
Ab 3698/cis-
5−BSA
compound
trans-DCCA-glycine
cis-DCCA-glycine
trans-DCCA
permethrin
glycine
PBA-glycine
1.9 (100)
6.7 (28)
430 (0.40)
2.6 (100)
18 (15)
808 (0.30)
0.8 (100)
37 (2)
>8200 (<0.01)
2357 (0.04)
>8200 (<0.01)
>8200 (<0.01)
4475 (0.04)
>19 000 (<0.01)
>19 000 (<0.01)
2357 (0.11)
>26 000 (<0.01)
>26 000 (<0.01)
IC (CR [%])
50
Ab 3701/trans-
5−BSA
Ab 3709/trans-
5−BSA
Ab 369/trans-
5−BSA
ABBREVIATIONS USED
cis-DCCA-glycine
trans-DCCA-glycine
cis-DCCA
permethrin
glycine
3.1 (100)
194 (1.61)
590 (0.53)
0.7 (100)
179 (0.38)
22 (3.00)
>6700 (<0.01)
>6700 (<0.01)
>6700 (<0.01)
6.2 (100)
322 (1.93)
2947 (0.21)
>61 900 (<0.01)
>61 900 (<0.01)
>61 900 (<0.01)
Ab, antibody; Thyr, thyroglobulin; BSA, bovine serum
albumin; DMF, N,N-dimethylformamide; DMSO, dimethyl
sulfoxide; ELISA, enzyme-linked immunosorbent assay; FAB-
MS, fast atom bombardment mass spectrum; GAR-HRP, goat
anti-rabbit immunoglobulin conjugated to horseradish peroxi-
dase; IC50, concentration of analyte giving 50% inhibition; sulfo-
NHS, N-hydroxysulfosuccinimide sodium salt; OD, optical
density; PBST, phosphate-buffered saline with 0.05% of Tween
20; TMB, tetramethylbenzidine; DCCA, 3-(2,2-dichlorovinyl)-
2,2-dimethylcyclopropane-1-carboxylic acid; DCCA-glycine,
N-(3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carbonyl)-
glycine; PBOH, 3-phenoxybenzyl alcohol; PBA, 3-phenoxy-
benzoic acid.
>31 400 (<0.01)
>31 400 (<0.01)
>31 400 (<0.01)
PBA-glycine
a The antibodies, Ab 3703, Ab 3710, and Ab 3698, were produced against the
trans configuration of immunogens, and the antibodies, Ab 3701, Ab 3709, and
Ab 369, were produced against the cis configuration of immunogens. The
compounds were prepared in the PBS solution.
(Ab 3710 and 3698). The ELISA combined with Ab 3703 and
coating antigen cis-hapten 5-BSA can be good for the detection
of total cis/trans-DCCA-glycine. All of the antibodies showed
no or very little CRs to free permethrin metabolite (DCCA),
permethrin, PBA-glycine conjugate, and glycine, suggesting that
the antibodies are very selective for the target structure.
Unlike the above antibodies produced against the trans-
immunogens, the antibodies, Ab 3701, Ab 3709, and Ab 369,
generated against the cis-configuration, had a low CR to the
trans-configuration. The antibodies showed a high selectivity
for the cis-target analyte. These antibodies also showed very
low CRs to other structurally related compounds. On the basis
of these results, it is thought that the most antigenic determinants
of the target analyte for the Ab production are the glycine moiety
(-NHCH2COO-), and the cis- or trans-isomeric configuration
of the structure.
ACKNOWLEDGMENT
We thank Dr. Donald W. Stoutamire for supplying cis- and
trans-DCCA as the starting material for the hapten synthesis,
Dr. In Hae Kim for advice on the synthesis, and the Department
of Chemistry (University of California, Davis) for the use of
its NMR instrument.
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Received for review March 3, 2004. Revised manuscript received May
5, 2004. Accepted May 10, 2004. This research has been supported in
part by the NIEHS Superfund Basic Research Program P42 ES04699,
the NIEHS Center for Environmental Health Sciences P30 ES05707,
the Department of Defense U.S. Army Medical Research and Material
Command contract DAMD17-01-1-0769, and the NIOSH Center for
Agricultural Disease and Research, Education and Prevention 1 U50
OH07550.
JF049646R