Development of an Enzyme-Linked Immunosorbent Assay for the Detection of the Pyrethroid Insecticide Fenpropathrin

Article abstract of DOI:10.1021/jf9710847

A competitive enayme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of fenpropathrin [(RS)-α-cyano-3-phenoxybenzyl-2,2,3,3-tetramethylcyclopropanecarboxylate]. Polyclonal antisera were isolated from rabbits immunized with two different fenpropathrin hapten conjugates. One hapten contained an amino function; the other contained a carboxyl group for conjugation to carrier proteins. Mollusk hemocyanins, thyroglobulin, and fetuin were used as carrier proteins. The antisera varied greatly in their affinities for fenpropathrin. A homologous assay system using the coating antigen format was the most sensitive. The IC⁵⁰ for fenpropathrin was 20 μg/L, and the lower detection limit was 2.5 μg/L. Pyrethroids, such as phenothrin, permethrin, resmethrin, fenvalerate, deltamethrin, cyfluthrin, and cypermethrin, and the pyrethroid metabolites, 3-phenoxybenzoic acid and fenpropathrin acid, did not cross-react significantly in this assay. Ten percent acetone or methanol and a pH of 4 were determined to be optimum assay conditions. Various cationic, anionic, and nonionic detergents had no significant effect on the assay.

Full text of DOI:10.1021/jf9710847

J. Agric. Food Chem. 1998, 46, 2211−2221
2211
Develop m en t of a n En zym e-Lin k ed Im m u n osor ben t Assa y for th e
Detection of th e P yr eth r oid In secticid e F en p r op a th r in
Ingrid Wengatz,† Donald W. Stoutamire, Shirley J . Gee, and Bruce D. Hammock*
Departments of Entomology and Environmental Toxicology, University of California,
Davis, California 95616
A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative
detection of fenpropathrin [(RS)-R-cyano-3-phenoxybenzyl-2,2,3,3-tetramethylcyclopropanecarbox-
ylate]. Polyclonal antisera were isolated from rabbits immunized with two different fenpropathrin
hapten conjugates. One hapten contained an amino function; the other contained a carboxyl group
for conjugation to carrier proteins. Mollusk hemocyanins, thyroglobulin, and fetuin were used as
carrier proteins. The antisera varied greatly in their affinities for fenpropathrin. A homologous
assay system using the coating antigen format was the most sensitive. The IC50 for fenpropathrin
was 20 µg/L, and the lower detection limit was 2.5 µg/L. Pyrethroids, such as phenothrin, permethrin,
resmethrin, fenvalerate, deltamethrin, cyfluthrin, and cypermethrin, and the pyrethroid metabolites,
3-phenoxybenzoic acid and fenpropathrin acid, did not cross-react significantly in this assay. Ten
percent acetone or methanol and a pH of 4 were determined to be optimum assay conditions. Various
cationic, anionic, and nonionic detergents had no significant effect on the assay.
Keyw or d s: Fenpropathrin; pyrethroid; ELISA; pesticide; enzyme immunoassay; cross-reactivity
INTRODUCTION
Fenpropathrin (Figure 1), commercial name Danitol
or Meothrin, is a synthetic pyrethroid insecticide, which
is more photostable (Leahey, 1985) than the naturally
occurring pyrethrum from which the synthetic pyre-
throids were designed. Fenpropathrin is predominantly
used to control various insects and mites that infest fruit
plants, vegetables, and other crops. In the United
States, as much as 28 800 kg of active ingredient is
applied annually for crop protection (Giannessi and
Anderson, 1995). Many pyrethroids are highly potent
insecticides with relatively low toxicity to mammals
(oral LD50 to female mice is 58 mg/kg; Fujita, 1981) and
most nontarget organisms (Elliott, 1977). However,
some nontarget organisms tested, such as fish, showed
significant toxicity (LC50 for bluegill sunfish of 1.95 µg/
L, 48 h; Tomlin, 1994). Thus, there is an interest in
monitoring levels of pyrethroids in aquatic ecosystems.
Multistep sample cleanup procedures are used in
conventional pyrethroid residue analysis followed by gas
chromatography (GC) (Blass, 1990; Takimoto et al.,
1984; Baker and Bottomley, 1982) or high-pressure
liquid chromatography (HPLC) (Sakaue et al., 1982).
Immunoassays are highly sensitive and selective ana-
lytical tools for detecting trace amounts of chemicals
such as pesticides (Hammock et al., 1990; Meulenberg
et al., 1995). They also offer the advantage of decreased
sample preparation, in some cases resulting in increased
sample throughput.
F igu r e 1. Structure of fenpropathrin (1).
applicators and farm workers to pyrethroids, such as
fenpropathrin, to reduce occupational exposure (Chen
et al., 1991). Another possible application of immu-
noassays for pyrethroid residue analysis is the monitor-
ing of food (Stanker et al., 1989; Skerritt et al., 1992;
Hill et al., 1993) and environmental samples (Bonwick
et al., 1994). The expected residues in food are generally
low because of low application rates and relatively rapid
degradation in the environment. Monitoring the aquatic
environment and associated sediments for fenpropath-
rin content is equally important because of its long half-
life (11-8520 days at 25 °C) in natural waters (Taka-
hashi et al., 1985) and its relative toxicity to nontarget
aquatic organisms.
One of the first immunoassays developed for a pyre-
throid, S-bioallethrin (Wing et al., 1978), utilized anti-
bodies that were stereoselective and could detect S-bio-
allethrin in the picomole range. Recently, monoclonal
antibodies for the detection of allethrin have been
developed and used in assays by Pullen and Hock
(1995). Assays have also been reported for permethrin
(Bonwick et al., 1994), bioresmethrin (Hill et al., 1993),
and phenothrin (Skerritt et al., 1992).
There are a variety of applications in which immu-
noassays may be advantageous. For example, one use
for immunoassays would be to monitor the exposure of
Challenges encountered in developing immunoassays
for pyrethroids lie in their lipophilicity and the synthesis
of appropriate haptens (Skerritt and Lee, 1996). Fen-
propathrin is very lipophilic (KOW of 100 000, 20 °C;
Tomlin, 1994), similar to other pyrethroids having a
phenoxybenzyl group. The very low water solubility of
fenpropathrin (14.1 µg/L at 25 °C) may cause yields from
* Author to whom correspondence should be addressed
[telephone (916) 752-7519; e-mail bdhammock@ucdavis.edu].
† Present address: EPA-NERL/Human Exposure Research
Branch, P.O. Box 93478, Las Vegas, NV 89193-3478.
S0021-8561(97)01084-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/08/1998

2212 J. Agric. Food Chem., Vol. 46, No. 6, 1998
Wengatz et al.
Sch em e 1
Spots were visualized under ultraviolet light or after staining
with iodine vapor. Flash chromatographic separations were
carried out on 40 µm average particle size Baker silica gel,
packed in glass columns of such diameter to give a column
height/diameter ratio of ≈7. Compounds were eluted using
the indicated solvents, where the f symbol denotes a stepwise
concentration gradient.
The coupling reagents 1-cyclohexyl-3-(2-morpholinoethyl)-
carbodiimide-metho-p-toluenesulfonate (morpho-CDI), 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(DAPEC), N-hydroxysuccinimide (NHS), and N-hydroxysul-
fosuccinimide (sulfo-NHS) were purchased from Aldrich. Presto
desalting plastic columns (5 mL volume) were obtained from
Pierce (Rockford, IL).
Anti-rabbit immunoglobulin, raised in goats and conjugated
to horseradish peroxidase (GAR/HRP), 3,3′,5,5′-tetramethyl-
benzidine (TMB), bovine serum albumin (BSA), ovalbumin
(OVA), hemocyanin from Limulus polyphemus (LPH), hemocy-
anin from keyhole limpet (KLH), thyroglobulin, and fetuin
were purchased from Sigma Chemical Co. (St. Louis, MO).
Microtiter plates 4-42404 were purchased from Nunc (Rosk-
ilde, Denmark).
In str u m en ts. NMR spectra were obtained using a General
Electric QE-300 spectrometer (Bruker NMR, Billerica, MA).
Chemical shift values are given in parts per million downfield
from internal tetramethylsilane. Melting points were deter-
mined on a Uni-Melt apparatus (Thomas Scientific, Swedes-
borogh, NJ ) and are uncorrected. Gas-liquid chromatograms
were determined on an HP 5890 GLC (Hewlett-Packard Corp.,
Avondale, PA) fitted with a 15 m, 0.32 mm i.d., capillary
column with a 0.25 µm film of dimethylpolysiloxane containing
5% of the methyl groups substituted by phenyl groups (J &W
Scientific, Folsom, CA). Fast atom bombardment high-resolu-
tion mass spectra (FAB-HRMS) were obtained on a ZAB-HS-
2F mass spectrometer (VG Analytical, Wythenshawe, U.K.),
using xenon (8 keV, 1 mA) for ionization and 3-nitrobenzyl
alcohol or glycerol as the matrix. Poly(ethylene glycol) was
added to the matrix as a mass calibrant. ELISAs were carried
out using 96-well microtiter plates and the absorbances read
with the Vmax reader from Molecular Devices (Menlo Park, CA).
Ha p ten Syn th esis a n d Ver ifica tion . Syntheses of the
haptens were carried out as outlined in Schemes 1 and 2.
Analytical data verifying the structures are provided. Racemic
fenpropathrin standard was prepared from 2,2,3,3-tetrameth-
the primarily aqueous conjugations to be low. The
lipophilicity may also affect the ELISA format develop-
ment, due to nonspecific binding of the lipophilic
molecule to the surface of working materials such as
microtiter plates or glass vials.
To our knowledge no work has been published on the
development of immunoassays to fenpropathrin. Strat-
egies for hapten synthesis for assays of other pyre-
throids containing an ester of an aryl cyanohydrin have
been comprehensively reviewed (Skerritt and Lee, 1996).
Since antibodies bind to the portion of the molecule
distal to the attachment site to the carrier protein,
pyrethroid haptens have been developed that link the
hapten to the protein (1) through the 3-phenoxybenzyl
end of the molecule, (2) through the R-cyano moiety of
the molecule, and (3) through fragments of the molecule
such as metabolites. More selective assays would be
developed from haptens made from the whole molecule.
Thus, we opted to couple the hapten through the
3-phenoxybenzyl end of the fenpropathrin molecule. One
of the haptens used was the same as reported by
Demoute and Touer (1987; an alkyl side chain termi-
nated by a carboxylic acid; hapten 6, Scheme 1) but
synthesized by a different route. The other was a
unique hapten containing an amine group on the
3-phenoxybenzyl moiety (hapten 8, Scheme 1). This
paper describes the development of the immunoassay
resulting from the above haptens.
MATERIALS AND METHODS
Ch em ica ls a n d Im m u n oa ssa y Rea gen ts. The pyrethroid
standards of fenvalerate, deltamethrin, phenothrin, per-
methrin, cypermethrin, cyfluthrin, and resmethrin were ob-
tained from Riedel de Haen (Seelze, Germany). Racemic
fenpropathrin was synthesized as described below with a
purity of g99% based on analytical data. Other organic
starting materials for hapten synthesis were obtained from
Aldrich Chemical Co. (Milwaukee, WI). For thin-layer chro-
matography (TLC), 0.2 mm precoated silica gel 60 F254 on
glass plates from E. Merck (Darmstadt, Germany) was used.

Development of a Fenpropathrin ELISA
J. Agric. Food Chem., Vol. 46, No. 6, 1998 2213
Sch em e 2
was continued for 10 h to collect 1.48 mL of water. Excess
benzyl alcohol was removed under vacuum (1 Torr) to a kettle
temperature of 150 °C. The residue was dissolved in ether,
washed with sodium bicarbonate solution, followed by a water
wash, and dried (MgSO4). The stripped residue was distilled
through a short-path head from an oil bath at 230-240 °C to
collect 20 g (91%) of product at a head temperature of 185-
194 °C (0.08 Torr), which displayed only one spot by TLC
1
(ether/CH2Cl2, 1:9): Rf 0.47; H NMR (CDCl3) δ 2.64 (t, J )
7.8 Hz, 2 H, CH2), 2.89 (t, 2 H, J ) 7.7 Hz, CH2), 5.1 (s, 2 H,
CH2Ar), 5.5 (s, 1 H, OH), 6.73 (d, J ) 8.4 Hz, 2 H), 7.03 (d, J
) 8.4 Hz, 2 H), 7.27-7.33 (m, 5 H, Ar).
Benzyl 4-(3-Formylphenoxy)benzenepropanoate (4). Potas-
sium tert-butoxide (65 mL, 1 M in tert-butyl alcohol) was added
under N2 to a stirred solution of benzyl 4-hydroxybenzenepro-
panoate (3) (16.6 g, 64.8 mmol), in 180 mL of xylene, and the
mixture was heated to remove 100 mL of distillate. An
additional 40 mL of xylene was added and distilled to a kettle
temperature of 136 °C. The resulting salt suspension was
cooled, treated with pyridine (16 mL), cuprous chloride (0.6
g), copper powder (0.3 g), 18-crown-6 (100 mg), and 15.0 g (65
mmol) of 1-bromo-3-(dimethoxymethyl) benzene (2). This
mixture was refluxed under N2 for 17 h, cooled, washed with
water, and filtered through silica gel (25 g). The pad of silica
gel was washed with CH2Cl2 (40 mL). The combined filtrate
and washing was evaporated to a volume of 30 mL, diluted
with wet CH2Cl2 (40 mL), and treated with acidified silica gel
(30 g, previously treated in ether with 9 drops of 98% H2SO4
and stripped to a dry powder on a rotary evaporator). Water
(1 mL) was added, and this mixture was stirred for 2 h. After
filtration (30 mL) and evaporation, the mixture was flash
chromatographed on silica gel (200 g), eluting with hexane/
CH2Cl2 (80:20 f 0:100). Stripping at 30 °C and 0.1 Torr
yielded 13.1 g (56%) of 4, which was a pale yellow oil showing
ylcyclopropanecarboxylic acid and 3-phenoxybenzaldehyde cy-
anohydrin and assigned a purity of g99% according to TLC,
NMR, and GLC analyses. All intermediates and haptens were
synthesized as racemic mixtures.
Fenpropathrin (1). Thionyl chloride (2.1 mL, 29 mmol) was
added to a mixture of 2,2,3,3-tetramethylcyclopropanecar-
boxylic acid (3.0 g, 21 mmol) and 1 µL of DMF in 9 mL of
CHCl3. The mixture was stirred and heated in an oil bath at
60-65 °C for 95 min. The mixture was stripped, hexane was
added to the residue, and the solution was evaporated to yield
the acid chloride, which was a colorless oil. 3-Phenoxybenz-
aldehyde (4.18 g, 21.1 mmol) and potassium cyanide (2.1 g,
32 mmol) in 9 mL of tetrahydrofuran (THF) and 0.9 mL of
water was stirred with cooling as 2.26 mL of 12.1 N HCl was
injected (Caution! Excess HCN generated!). After a mild
exotherm, the mixture was stirred at ambient temperature for
1 h, diluted with ether, acidified with 3 N HCl, washed three
times with water, and stripped of solvent. Hexane was added,
and the mixture was restripped to yield the cyanohydrin,
which was an oil. This was dissolved in 15 mL of CH2Cl2 and
stirred with ice cooling as the above acid chloride, in 15 mL of
the same solvent, was added all at once followed immediately
by 2.2 mL of pyridine. After 0.5 h, the mixture was washed
twice with water, dried (MgSO4), and stripped. Flash chro-
matography on 80 g of silica gel (hexane f CH2Cl2) yielded a
product that was contaminated with 2,2,3,3-tetramethylcyclo-
propanecarboxylic acid. This acid was removed by extraction
from hexane solution with aqueous sodium bicarbonate solu-
tion. The resulting solution of fenpropathrin was water
washed, stripped, and distilled through a Kugelrohr apparatus
at 150-160 °C (0.07 Torr) to yield 4.8 g, 65%, of 1, which
showed only one spot by TLC (CH2Cl2): Rf 0.63; 1H NMR
(CDCl3) δ 1.18 (s, 3 H, CH3), 1.21 (s, 3 H, CH3), 1.22 (s, 3 H,
CH3) 1.26 (s, 1 H, CHCO2), 1.27 (s, 3 H, CH3), 6.35 (s, 1H,
CHCN), 7.01-7.42 (m, 9 H, Ar); 13C NMR (CDCl3) δ 16.4 (2
CH3), 23.3 (2 CH3), 31.9, 32.1, 35.0, 61.5 (CCN), 116.4 (CN),
117.5, 119.2 (2 CH), 119.8, 121.9, 123.9, 129.9 (2 CH), 130.4,
134.1, 156.2, 158.0, 169.7 (CdO). GLC analysis indicated a
purity of >99%.
1-Bromo-3-(dimethoxymethyl)benzene (2). This product was
prepared using a modification of literature procedures (Young
et al., 1980; Creary and Aldridge, 1991). Two drops of
concentrated sulfuric acid was added to a solution of 3-bro-
mobenzaldehyde (18.5 g, 0.1 mol) and trimethyl orthoformate
(12.2 g, 0.115 mol) in 15 mL of methanol. After an immediate
mild exotherm and 3 h at ambient temperature, the mixture
was diluted with ether, washed with a sodium carbonate
solution, followed by a water wash, dried (MgSO4), stripped,
and vacuum distilled to yield 22 g, 95%, of 2, which was a
colorless oil: bp 60-63 °C (0.05 Torr); 1H NMR (CDCl3) δ 3.32
(s, 6 H, CH3), 5.36 (s, 1 H), 7.21-7.63 (m, 4 H, Ar).
Benzyl 4-Hydroxybenzenepropanoate (3). A mixture of 4-hy-
droxybenzenepropanoic acid (14.4 g, 86.6 mmol), benzyl alcohol
(27 mL), toluene (15 mL), and 4 drops of 85% phosphoric acid
was heated to reflux under a Dean-Stark trap. Toluene was
removed up to a kettle temperature of 145-150 °C. Heating
1
only one spot by TLC (CH2Cl2): Rf 0.36; H NMR (CDCl3) δ
2.69 (t, J ) 8.0 Hz, 2 H, CH2), 2.97 (t, J ) 7.8 Hz, 2 H, CH2),
5.12 (s, 2 H, CH2Ar), 6.92-7.57 (m, 13 H, Ar), 9.94 (s, 1 H,
CHO); 13C NMR (CDCl3) δ 30.0 (CH2), 35.7 (CH2CO), 66.1 (CH2-
Ar), 117.7, 119.4 (2 C), 124.1, 124.3, 128.0 (3 C), 128.4 (2 C),
129.7 (2 C), 130.2, 135.8, 136.2, 137.9, 154.3, 158.4, 172.3
(COOR), 191.3 (CHO).
Cya no-[3-[4-[(3-oxo-3-benzyloxy)propyl]phenoxy]phenyl]-
methyl 2,2,3,3-Tetramethylcyclopropanecarboxylate (5). The
aldehyde 4 (0.75 g, 2.1 mmol), in 1 mL of THF and 0.2 mL of
H2O, was cooled in ice and treated with powdered KCN (203
mg, 3.1 mmol) followed by 0.215 mL of 12.1 N HCl (Caution!
Excess HCN generated!). The reaction was mildly exothermic.
After stirring for 30 min, the mixture was acidified with 3 N
HCl and extracted with ether, and the organic phase was
washed with water, dried (MgSO4), and stripped to give the
cyanohydrin, which was a brown oil. The cyanohydrin, in 1
mL of CH2Cl2, was stirred and cooled in ice and treated with
2,2,3,3-tetramethylcyclopropanecarbonyl chloride (prepared as
described from 311 mg of the acid above) in 1 mL of CH2Cl2,
and pyridine (0.23 mL) was injected immediately. After 10
min of ice cooling, the mixture was stirred for 20 min at
ambient temperature, washed twice with water, and stripped
to yield a brown gum. Chromatography on silica gel (hexane
f CH2Cl2) gave the pure ester, 0.71 g (88%), which was a
colorless gum: 1H NMR (CDCl3) δ 1.17 (s, 3 H, CH3), 1.21 (s,
3 H, CH3), 1.22 (s, 3 H, CH3), 1.24 (s, 1 H, CHCO), 1.25 (s, 3
H, CH3), 2.68 (t, J ) 7.7 Hz, 2 H, CH2), 2.96 (t, J ) 7.7 Hz, 2
H, CH2), 5.12 (s, 2 H, CH2Ar), 6.34 (s, 1 H, CHCN), 6.9-7.4
(m, 13 H, Ar).
4-[3-(2,2,3,3-Tetramethylcyclopropane-1-carbonyloxy(cyano)-
methyl)phenoxy]benzenepropanoic Acid (6). The ester 5 (0.600
g, 1.17 mmol) in 1.5 mL of dry CH2Cl2 was treated with 20 µL
of bis(trimethylsilyl)trifluoroacetamide (BSTFA) followed by
iodotrimethylsilane (TMSI, 0.175 mL). After 1.5 h, 5 drops of
water and 1 mL of CH2Cl2 were added and the mixture was
stirred for 5 min. The organic phase was immediately flash
chromatographed on 10 g of silica gel (CH2Cl2 f ethyl acetate).

2214 J. Agric. Food Chem., Vol. 46, No. 6, 1998
Wengatz et al.
The product was stripped (<1 mm) to yield 0.47 g of 6, which
was a pale yellow gum: 1H NMR (CDCl3) δ 1.18 (s, 3 H, CH3),
1.21 (s, 3 H, CH3), 1.22 (s, 3 H, CH3) 1.26 (s, 1 H, CHCO), 1.27
(s, 3 H, CH3), 2.69 (t, J ) 7.7 Hz, 2 H, CH2), 2.96 (t, J ) 7.7
Hz, 2 H, CH2), 6.34 (s, 1 H, CHCN), 6.94-7.42 (m, 8 H, Ar);
13C NMR (CDCl3) δ 16.4 (2 CH3), 23.3 (2 CH3), 29.8 (CH2), 31.9
and 32.1 (cyclopropyl), 35.0 (CH2), 35.6, 61.6, 116.4 (CN), 117.4,
119.4 (2 C), 119.6, 121.9, 129.7 (2 C), 130.4, 134.0, 135.8, 154.7,
158.1, 169.8, (COO), 178.9 (COOH). This product was con-
taminated with ∼10% of compound 6b, the tetramethylcyclo-
propanecarboxylic acid esterified with 4-hydroxybenzenepro-
pionic acid, as determined by GLC of the methyl ester
(prepared via trimethylsilyldiazomethane). Compound 6b was
synthesized via 3 in 70% yield from 6a in the same way and
was obtained as a white solid: mp 139-141 °C; 1H NMR-
(CDCl3) δ 1.26 (s, 6 H, 2 CH3), 1.28 (s, 6 H, 2 CH3), 1.42 (s, 1
H, CH), 2.66 (t, J ) 7.7 Hz, 2 H, CH2), 2.94 (t, J ) 7.7 Hz, 2
H, CH2), 7.00 (d, J ) 8.5 Hz, 2 H, Ar), 7.20 (d, J ) 8.5 Hz, 2
H, Ar). The Rf values of this compound, in a variety of solvent
systems (2% acetic acid in isopropyl alcohol/ethyl acetate, 1:2,
Rf 0.71; 1.5% acetic acid in ethyl acetate/hexane, 1:1, Rf 0.46;
1% acetic acid in hexane/ether, 1:1, Rf 0.27; 0.3% acetic acid
in ethyl acetate/nutyl chloride, 1:9, Rf 0.12), were all identical
with those of 6, which made the purification by flash chroma-
tography difficult. Only multiple development of the TLC
plate using 0.3% acetic acid in ethyl acetate/hexane, 1:9, Rf
0.01-0.02 (first development), resulted in separation.
Cyano[3-(4-nitrophenoxy)phenyl]methyl-2,2,3,3-tetramethyl-
cyclopropanecarboxylate (4-Nitrofenpropathrin) (7). The cy-
anohydrin of 3-(4-nitrophenoxy)benzaldehyde (Loewe and
Urbanietz, 1967) was prepared (2.0 mmol), following the
procedure described above (Caution! Excess HCN generated!).
It was dissolved in 2 mL of chilled CH2Cl2 and added all at
once to a stirred ice-cooled solution of 2,2,3,3-tetramethylcy-
clopropanecarbonyl chloride (2 mmol) in 2 mL of CH2Cl2
prepared as described above. Pyridine (0.18 mL, 2.2 mmol)
was immediately added with stirring. After an immediate
mild exotherm and stirring at ambient temperature for 2 h,
the mixture was acidified with 3 N HCl, washed twice with
water, filtered through 3 g of silica gel, followed by 25 mL of
CH2Cl2, and stripped to yield a yellow oil. Flash chromatog-
raphy on 20 g of silica gel (20 f 80% CH2Cl2 in hexane) yielded
0.60 g (47%) of 7, which was a pure colorless gum, plus an
additional 0.45 g (34%) that contained a trace of the starting
aldehyde: TLC (CH2Cl2), Rf 0.4; 1H NMR (CDCl3) δ 1.19 (s, 3
H, CH3), 1.22 (s, 3 H, CH3), 1.24 (s, 3 H, CH3), 1.27 [s, 1 H,
CHC(O)], 1.28 (s, 3 H, CH3), 6.40 (s, 1 H, CHCN), 7.06 and
8.24 (d’s, J ) 9.2 Hz, 4 H, NO2Ar), 7.0-7.6 (m, 4 H, Ar).
Cyano[3-(4-aminophenoxy)phenyl]methyl-2,2,3,3-tetrameth-
ylcyclopropanecarboxylate (4-Aminofenpropathrin) (8). Stan-
nous chloride dihydrate (0.85 g, 3.7 mmol) was added under
N2 to a stirred solution of the nitroester 7 (298 mg, 0.75 mmol)
in 3 mL of ethanol. The mixture was heated at 70 °C for 35
min and poured into water (10 mL) containing 0.7 g of Celite
and 0.72 g of KHCO3. Filtration, followed by extraction of
solids with ethyl acetate and stripping of the solvent, yielded
a red gum. Flash chromatography on silica gel (hexane f CH2-
Cl2 f ether) yielded 12 mg of starting ester and 190 mg (69%)
of 8, which was a pale yellow gum: TLC (CH2Cl2), Rf 0.24; 1H
NMR (CDCl3) δ 1.18 (s, 3 H, CH3), 1.21 (s, 3 H, CH3), 1.22 (s,
3 H, CH3), 1.25 [s, 1 H, CHC(O)], 1.27 (s’s, 3 H, CH3), 3.65 (b,
2 H, NH2), 6.31(s, 1 H, CHCN), 6.70 and 6.88 (d’s, J ) 4.4 Hz,
4 H, N-C6H4), 6.68-7.40 (m, 4 H, Ar); FAB-HRMS m/z calcd
for C22H24N2O3 364.1784, observed 364.1767.
2.38 (t, J ) 7.4 Hz, 2 H, CH2), 3.39 (t, J ) 6.8 Hz, 2 H, CH2),
5.12 (s, 2 H, CH2Ar), 7.32-7.36 (m, 5 H, Ar).
Benzyl 2-(3-Formylphenoxy)acetate (10). Potassium tert-
butoxide (10.1 g, 90 mmol) was added with cooling under N2
to a stirred solution of 3-hydroxybenzaldehyde (11.0 g, 90
mmol) in 70 mL of dimethyl sulfoxide (DMSO). Benzyl
bromoacetate (19.6 g, 86 mmol) was added over ∼5 min. After
2.5 h at ambient temperature, the mixture was diluted with
water (200 mL) and extracted with hexane. The hexane
solution was washed with water and stripped of solvent to yield
a yellow oil. Flash chromatography on 200 g of silica gel (n-
BuCl f CH2Cl2) and vacuum stripping of fractions containing
pure product yielded 18.2 g, (75%) of 10, which was a light
yellow oil: 1H NMR (CDCl3) δ 4.74 (s, 2 H, CH2CO), 5.25 (s, 2
H, CH2Ar), 7.2-7.53 (m, 9 H, Ar), 9.94 (s, 1 H, CHO).
Benzyl 6-(3-Formylphenoxy)hexanoate (11). A solution of
3-hydroxybenzaldehyde (1.88 g, 15.4 mmol) in 30 mL of DMSO
was stirred under N2 and treated with 17 mL of a 1 M solution
of potassium tert-butoxide in tert-butyl alcohol followed im-
mediately by addition of the ester 9 (4.0 g, 14 mmol). After
stirring at 25 °C for 10 h, the mixture was diluted with water,
acidified with dilute HCl, and extracted with ether/hexane.
The extract was water washed and stripped. Flash chroma-
tography of the residue on silica gel (BuCl f CH2Cl2 f ethyl
acetate) yielded 4.0 g (87%) of 11, which was a colorless oil:
1H NMR (CDCl3) δ 1.52 (m, 2 H, CH2), 1.73 (quin, J ) 7.8 Hz,
2 H, CH2), 1.82 (quin, J ) 7.4 Hz, 2 H, CH2), 2.40 (t, J ) 7.4
Hz, 2 H, CH2), 4.00 (t, J ) 6.4 Hz, 2 H, CH2), 5.12 (s, 2 H,
CH2Ar), 7.13-7.43 (m, 9 H, Ar), 9.97 (s, 1 H, CHO).
Benzyl 3-[Cyano-(2,2,3,3-tetramethylcyclopropanecarbonyloxy)-
methyl]phenoxyacetate (12). The aldehyde 11 (2.0 g, 7.4 mmol)
was converted to the cyanohydrin according to the same
procedure as for the cyanohydrin of 4 described above (Cau-
tion! Excess HCN generated!). This was reacted with 2,2,3,3-
tetramethylcyclopropanecarbonyl chloride using the same
procedure described above for 5. Flash chromatography on
90 g of silica gel (n-BuCl f CH2Cl2) yielded 2.6 g (83%) of 12,
which was a colorless oil: 1H NMR (CDCl3) δ 1.18 (s, 3 H, CH3),
1.23 (s, 6 H, 2 CH3), 1.25 [s, 1 H, CHC(O)], 1.28 (s, 3 H, CH3),
4.69 [s, 2 H, CH2C(O)], 5.24 (s, 2 H, CH2Ar), 6.33 (s, 1 H,
CHCN), 6.94-7.38 (m, 9 H, Ar).
3-[Cyano-(2,2,3,3-tetramethylcyclopropanecarbonyloxy)methyl]-
phenoxyacetic Acid (13). The ester 12 (0.75 g, 1.78 mmol) in
1.8 mL of CH2Cl2 was treated under N2 with 20 µL of BSTFA
followed by 0.266 mL of TMSI. After 18 h at ambient
temperature, the mixture was treated with 2 mL of methanol,
washed with dilute HCl, stripped, and flash chromatographed
on 20 g of silica gel (CH2Cl2 f 3% acetic acid/ethyl acetate).
Crystallization of the appropriate evaporated fractions from
CCl4 yielded 13 in three crops, which was a white solid totaling
1
480 mg (81%): mp 116.5-118.5 °C; H NMR (CDCl3) δ 1.18
(s, 3 H, CH3), 1.23 (s, 6 H, 2 CH3), 1.26 [s, 1 H, CHC(O)], 1.28
(s, 3 H, CH3), 4.72 (s, 2 H, CH2COOH), 6.32 (s, 1 H, CHCN),
6.97-7.41 (m, 4 H, Ar).
Benzyl 6-[3-(Cyano(2,2,3,3-tetramethylcyclopropanecarbon-
yloxy)methyl)phenoxy]hexanoate (14). The cyanohydrin of
aldehyde 11 was prepared on a 5.36 mmol scale as described
above for benzyl 2-(3-formylphenoxy)acetate (10). Reaction in
a similar manner with 2,2,3,3-tetramethylcyclopropanecarbo-
nyl chloride and flash chromatography of the product on 40 g
of silica gel (hexane f CH2Cl2) yielded 1.94 g (76%) of 14,
which was a colorless oil: 1H NMR (CDCl3) δ 1.17 (s, 3 H, CH3),
1.23 (s, 6 H, 2 CH3), 1.26 (s, 1 H, CHCO2), 1.28 (s, 3 H, CH3),
1.52 (m, 2 H, CH2), 1.77 (m, 4 H, 2 CH2), 2.41 (t, J ) 7.4 Hz,
2 H, CH2,), 3.96 (t, J ) 6.3 Hz, 2 H, CH2), 5.12 (s, 2 H, CH2-
Ar), 6.34 (s, 1 H, CHCN), 6.9-7.4 (m, 9 H, Ar).
6-[3-(Cyano(2,2,3,3-tetramethylcyclopropanecarbonyloxy)-
methyl)phenoxy]hexanoic Acid (15). The ester 14 (0.955 g, 2.00
mmol) in 2 mL of CH2Cl2 was treated with 20 µL of BSTFA
followed by TMSI (0.313 mL). After 18 h at ambient temper-
ature, some starting ester remained. The mixture was treated
with an additional 20 µL of BSTFA and 28 µL of TMSI for 4
h. Workup and flash chromatography on 25 g of silica gel (n-
BuCl f CH2Cl2 f ethyl acetate) recovered 21% of the starting
ester and the crude product. The product was crystallized in
Benzyl 6-Bromohexanoate (9). A mixture of 6-bromohex-
anoic acid (12.6 g, 65 mmol), benzyl alcohol (10 mL), 85%
phosphoric acid (10 drops), and 30 mL of benzene was heated
under a Dean-Stark trap with removal of solvent to a kettle
temperature of 135 °C. Boiling was continued for 3.5 h to
remove 1.38 mL of water. The reaction mixture was diluted
with hexane, washed with sodium bicarbonate solution fol-
lowed by water, and distilled through a short-path head to
yield 14.4 g (78%) of a colorless liquid: bp 129-145 °C (0.08
Torr); 1H NMR (CDCl3) δ 1.46 (m, J ) 7.4 Hz, 2 H, CH2), 1.67
(quin, J ) 7.6 Hz, 2 H, CH2), 1.86 (quin, J ) 7.1 Hz, 2 H, CH2),

Development of a Fenpropathrin ELISA
J. Agric. Food Chem., Vol. 46, No. 6, 1998 2215
Ta ble 1. Su m m a r y of Hom ologou s Assa y Com p on en tsa
a
Structures of the immunogens used to elicit antisera (AS) and the coating antigens are shown. The IC50 is listed for each assay
utilizing the respective coating antigen and antiserum combination.
two crops (n-BuCl/hexane) to yield 0.51 g of 15, which was a
white solid: mp 80-82.5 °C (84% based on recovered starting
Hapten Conjugates 8-Thyroglobulin and 8-BSA (Approach
B). Three vials, each containing a solution of hapten 8 (9 mg,
0.025 mmol) in 1.0 mL of 37.5 mM H2SO4 in DMSO, were
cooled to 16 °C. Butyl nitrite (100 µL of 0.375 M butyl nitrite
in DMSO) was added dropwise with stirring. Stirring was
continued for 10 min. The contents of one vial was added to
a solution of BSA, the other to a solution of thyroglobulin, and
the third to a solution of LPH. Each protein (20 mg) was
dissolved in 5 mL of borate buffer (0.1 M, pH 9.4). The stirring
was continued overnight at 4 °C. Each yellow reaction mixture
was purified by size exclusion chromatography, using a 5 mL
dextran desalting column and phosphate-buffered saline (PBS;
pH 7.5) solution as the eluant. Column fractions containing
purified conjugates were orange. These were stored as de-
scribed above.
Hapten Conjugate 8-Fetuin. To a stirred solution of fetuin
(12 mg, 0.002 mmol in 2 mL of PBS) was added dropwise
aqueous NaIO4 (200 µL of a 100 mM solution). Stirring was
continued for 20 min at room temperature. The solution was
dialyzed overnight at 4 °C against 1 mM sodium acetate buffer
(pH 4.4). This dialysate was transferred into a vial, and 450
µL of a 200 mM carbonate buffer (pH 9.5) was added, followed
by 6.2 mg (0.015 mmol) of hapten 8 in 2 mL of THF. This
mixture was stirred for 4.5 h at room temperature. Finally,
100 µL of freshly prepared NaBH4 solution (4 mg/mL water)
was added, and the solution was incubated for 2 h at room
temperature. The reaction mixture was purified by using a
dextran desalting column and PBS (pH 7.5) solution as the
eluant. The conjugate was stored as described above.
Hapten Conjugates 13-BSA and 13-OVA. To 19.8 mg (0.05
mmol) of hapten 13 in 2 mL of dry DMF were added 12.0 mg
(0.10 mmol) of NHS and 13.6 mg (0.060 mmol) of DAPEC. The
mixture was stirred overnight at ambient temperature and
divided into two equal aliquots. One aliquot was added to a
solution of BSA, the other one to a solution of OVA. Each
protein solution (10 mg) was dissolved in 7 mL of PBS. The
reaction mixtures were stirred for 1 h at ambient temperature
followed by 5 h at 4 °C, purified using dextran desalting
columns, and stored as described above.
Hapten Conjugates 15-BSA and 15-OVA. To 8.3 mg (0.025
mmol) of hapten 15 in 2 mL of dry DMF were added 9.0 mg
(0.040 mmol) of sulfo-NHS and 5.8 mg (0.030 mmol) of DAPEC
(Bekheit et al., 1993). The mixture was stirred overnight at
ambient temperature and then divided into two equal aliquots.
One aliquot was added to a solution of BSA, the other to a
1
material); H NMR (CDCl3) δ 1.18 (s, 3 H, CH3), 1.23 (s, 6 H,
2 CH3), 1.26 (s,1 H, CHCO2), 1.28 (s, 3 H, CH3), 1.54 (m, 2 H,
CH2), 1.73 (quin, J ) 7.5 Hz, 2 H, CH2), 1.83 (quin, J ) 7.1
Hz, 2 H, CH2), 2.41 (t, J ) 7.4 Hz, 2 H, CH2CO2), 3.98 (t, J )
6.3 Hz, 2 H, CH2O), 6.35 (s, 1 H, CHCN), 6.93-7.36 (m, 4 H,
Ar).
Ha p ten Con ju ga tion . Four different conjugation methods
were utilizedsthe water soluble carbodiimide, diazotization,
periodate, and activated ester methods (Tijssen, 1985; Er-
langer, 1973). To obtain immunogens, hapten 6 was conju-
gated to KLH, and hapten 8 was conjugated to the carrier
proteins LPH, thyroglobulin, and fetuin. Coating antigens
were made by coupling haptens 6, 8, 13, and 15 to BSA and
OVA (Table 1).
Hapten Conjugates 6-KLH and 6-BSA. Morpho-CDI (50
mg, 0.118 mmol) was added to a solution of 23.6 mg (0.056
mmol) of hapten 6 in 0.5 mL of dimethylformamide (DMF).
The solution was stirred for 45 min at ambient temperature.
DMF (1 mL) and 0.6 mL of H2O were added. The solution
was divided into two equal aliquots. One aliquot was added
to a solution of BSA, the other to a solution of KLH. Each
protein (100 mg) was dissolved in 16 mL of cold distilled water
(pH 6.5) plus 0.75 mL of DMF. The reaction mixture was
stirred for 2 h and purified by acetone precipitation. Ice-cold
acetone was added to the protein solution (1:1 v/v) and
centrifuged for 10 min at 3000g. Each precipitate was washed
three times with 5 mL of cold acetone, centrifuging between
each wash. The precipitate was resuspended in water and
stored in aliquots at -80, -20, and 4 °C.
Hapten Conjugates 8-LPH and 8-BSA (Approach A).
Sodium nitrite (0.5 mL of a 140 mg/10 mL water solution) was
added to hapten 8 (37.9 mg, 0.104 mmol) dissolved in 1 mL of
1 N HCl. The reaction vial was cooled with ice while the
solution was stirred. DMF (0.3 mL) was added and stirred
for 10 min, and the solution was divided into two equal
aliquots. One aliquot (0.9 mL) was added to a solution of BSA,
the other to a solution of LPH. The BSA (103 mg) and the
LPH (131 mg) were dissolved in 20 mL of ice-cold borate buffer
(0.2 M, pH 8.9). The reaction mixtures were cooled in an ice
bath and stirred continuously for 30 min. The pH of the yellow
solutions was adjusted to 7.0 with 1 N NaOH. Each mixture
was purified by acetone precipitation and stored as described
above.

2216 J. Agric. Food Chem., Vol. 46, No. 6, 1998
Wengatz et al.
Ta ble 2. Su m m a r y of Heter ologou s Assa y Com p on en tsa
a
Structures of the immunogens used to elicit antisera (AS) and the coating antigens are shown. The IC50 is listed for each assay
utilizing the respective coating antigen and antiserum combination.
Ta ble 3. Cr oss-Rea ctivities of P yr eth r oid s a n d Oth er
solution of OVA. Each protein (20 mg) was dissolved in 3 mL
Str u ctu r a lly Rela ted Com p ou n d sa
of PBS. Both reaction mixtures were stirred for 1.5 h at 4 °C,
followed by 7 h at ambient temperature, purified using dextran
desalting columns, and stored as described above.
cross-
cross-
compound
reactivityb
compound
reactivityb
fenpropathrin (1)
fenvalerate (16)
phenothrin (17)
permethrin (18)
cypermethrin (20)
resmethrin (19)
100.0
2.3
ni
ni
ni
deltamethrin (21)
DDT (23)
fenoxycarb (24)
chlorpropham (26)
3-phenoxybenzoic acid (22)
fenpropathrin acid (25)
18.8
nic
ni
16.3
ni
Im m u n iza tion a n d An tiser u m P r ep a r a tion . Each fe-
male New Zealand white rabbit was immunized intradermally
(Gee et al., 1988) with one of the immunogens listed in Tables
1 and 2. One month after an initial immunization with 100
µg of the immunogen protein dissolved in PBS and emulsified
with Freund’s complete adjuvant (1:1 v/v), further injections
of 100 µg of the immunogen that was emulsified with Freund’s
incomplete adjuvant were given. Booster injections were given
at 3 week intervals. The rabbits were bled 10 days after each
boost. After coagulation of the blood, the serum was isolated
after centrifugation for 10 min at 4 °C. Preimmune sera were
collected prior to immunization to provide control sera having
no related humoral immune response.
ni
ni
a
The assay consisted of coating antigen 8-BSA (1/8000) and
AS 58 (1/40000 dilution). All assay conditions were as described
b
under Materials and Methods. Cross-reactivity was calculated
as follows: (IC50 of fenpropathrin/IC50 of compound tested) × 100.
The IC50 of fenpropathrin was 16 µg/L. c ni, no inhibition at the
highest concentration tested (1000 µg/L).
for the immunogen were used. For standard curves, stock
solutions of pyrethroids in methanol or acetonitrile (10 mg/
2.5 mL) were diluted from 1 mg/L to 1 µg/L in 1:2 dilution
steps using 10% methanol in PBS as the diluent. The final
concentration of methanol in the well was 5%.
En zym e-Lin k ed Im m u n osor ben t Assa y (ELISA). The
coating antigen format was used in which BSA or OVA
conjugates were used as coating antigens. Each coating
antigen was diluted with coating buffer (100 mM carbonate-
bicarbonate buffer, pH 9.6). Microtiter plates were coated
overnight at 4 °C with 200 µL/well of the hapten-protein
conjugate. After the plates were washed with PBST (PBS with
Tween 20: 8 g/L NaCl, 1.15 g/L Na2HPO4, 0.2 g/L KH2PO4,
0.2 g/L KCl, and 0.05% Tween v/v), the surface of the wells
was blocked by adding a solution of OVA (1% in PBS, 200 µL/
well) and incubating for 30 min at room temperature. After
another washing step, 200 µL/well of antiserum diluted in PBS
(for determination of antibody titer) or 100 µL/well of antise-
rum diluted in PBS and 100 µL/well of standard solution were
dispensed into the wells and incubated for 1-2 h at room
temperature. Following another washing step (four times),
200 µL/well of GAR/HRP (diluted 1:8000 in PBS) was added
to each well and incubated for 1 h at room temperature. The
plate was washed four times, and 150 µL/well of substrate
solution (3.3 µL of 30% H2O2, 200 µL of 1.2% TMB in DMSO
per 25 mL of acetate buffer, pH 5.5) was pipetted into each
well. Ten to fifteen minutes later the color development was
stopped by adding 50 µL/well of 2 M H2SO4. The plates were
read in a dual-wavelength mode, subtracting the absorbance
at 650 nm from the absorbance at 450 nm. All experiments
were conducted using two or three well replicates.
Assa y Op tim iza tion . The following assay parameters
were investigated.
pH Effect. To vary the pH, the antibody was prepared in
phosphate buffer with pH values from 4.0 to 10.0. Analyte
was prepared as described above in 10% methanol in PBS. All
other assay conditions were as described above. The standard
curve was run in three well replicates.
Ionic Strength. The effect of PBS buffer with increasing
content of NaCl (0.1-1.0 M) was studied. As above, the
antibody was diluted in buffers of various ionic strengths, and
the assays were conducted as above.
Solvent Effect. The tolerance to various water-miscible
solvents used to dissolve pyrethroids (methanol, ethanol,
acetone, acetonitrile, and DMF) was tested. The analyte was
dissolved in solvents of various concentration levels (1, 2.5, 5,
7.5, 10, 20, and 40% in PBS). In this case, a fixed amount of
analyte (fenpropathrin) at the approximate IC50 level (20 µg/
L) was used in the ELISA. The control contained antibody
but no analyte.
Effect of Gelatin and Detergents as Additives. In one assay,
gelatin was added to the antiserum solution at six concentra-
tion levels (0.1-1.0%). In a second experiment, the antiserum
solution was prepared in several detergents. Neutral deter-
gents, Tween 20 (0.02%), PEG 3400 (0.02%), PEG 5000
(0.02%), and Triton X-100 (0.05%), were tested as well as
anionic detergents such as cholic acid (0.02%), deoxycholic acid
Both homologous and heterologous assays were assessed
(Tables 2 and 3). In the homologous assays the hapten
conjugation chemistry was the same for the immunogen and
the coating antigen. In the heterologous assays coating
antigens having coupling chemistry different from that used

Development of a Fenpropathrin ELISA
J. Agric. Food Chem., Vol. 46, No. 6, 1998 2217
(0.02%), SDS (sodium dodecyl sulfate 0.05%), glycodeoxycholic
acid (0.02%), dioctylsulfosuccinate (0.025% and 0.01%), and
3-(3-cholamidopropyl)dimethylamino-1-propanesulfate (0.02%)
and the cationic detergents dodecyltrimethylammonium bro-
mide (0.015%), dodecylethyldimethylammonium bromide
(0.015%), and Aliquat 336 (0.033%). These concentrations
were chosen because they were clearly below the critical
micellar concentrations of the detergents used.
changed to dark orange-brown on exposure to light, as
is typical of anilines.
In general, we chose to prepare haptens that contain
a functional group for coupling at the aromatic moiety
of the pyrethroid molecule. The cyclopropane moiety
of the molecule would be distal to the conjugation site,
and therefore it would present a primary target to elicit
compound-specific antibodies that have an affinity for
this part of the molecule. A set of immunogens was
synthesized using different haptens, containing amino
or carboxyl functionalities, and different carrier proteins
(see Tables 1 and 2).
For example, hapten 6 has two methylene groups
linking the carboxylic acid group to the aromatic portion
of the molecule. The conjugates were made using a
carbodiimide method (Tijssen, 1985). This method is
advantageous in that a lipophilic hapten is first dis-
solved in organic solvent. This serves to help keep it
in solution during further reaction with the protein in
the mostly aqueous medium. It was anticipated that
the short aliphatic spacer of the hapten would result in
an immunogen that would generate antibodies that also
bind to the aromatic moiety of the pyrethroid. In fact,
the antibodies generated (AS 6982) were able to bind
to a fenvalerate-based coating antigen (Table 1) that has
only the aromatic moiety in common with fenpropathrin.
In addition, the affinity of this antiserum for the
fenvalerate moiety is very high, since free fenpropathrin
was not easily able to displace the antibody from this
coating antigen, as evidenced by the high IC50.
Hapten 8 features an amino group in the para
position of the phenoxybenzyl group in contrast to the
carboxylic acid group of hapten 6. The aromatic amino
group was coupled to carrier proteins using a diazoti-
zation method (Tijssen, 1985). This method is advanta-
geous in that the reaction product is brightly colored,
making confirmation of the success of the reaction
immediate. In addition, the reactive intermediate is
water soluble, an advantage when working with lipo-
philic haptens. To synthesize immunogen and coating
antigens, diazo salts were made using NaNO2 under
acidic conditions (approach A). The resulting immuno-
gen, although highly colored, did not generate antibodies
that could be used in a competitive assay format. In
another approach (B) hapten 8 was coupled to the
carrier proteins by a modified diazotization reaction
using butyl nitrite in DMSO as described under Materi-
als and Methods. The antisera generated from this
immunogen (AS 56 and AS 58) were useful in both
homologous and heterologous assay formats (Tables 1
and 2).
Hapten 8 was also conjugated to bovine fetuin using
a sodium periodate method (Tijssen, 1985). Fetuin has
a molecular weight of ≈42 000 (Carr et al., 1993) with
an estimated carbohydrate content of 7% N-glycolyl-
neuraminic acid (Noguchi et al., 1995) consisting largely
of galactose, mannose, and glucose oligosaccharides
(Rice et al., 1990). Because it has been shown that an
increased carbohydrate content of the carrier increases
the antigenicity of the immunogen (Noguchi et al.,
1995), we used this glycoprotein as a carrier. Another
potential advantage is that the hydrophilic carbohydrate
moiety acts as a spacer between the lipophilic hapten
and the carrier surface, allowing the immunogen to
remain fairly hydrophilic despite attachment of a rather
lipophilic hapten. One rabbit was immunized with this
immunogen. The resulting antisera (AS 55) bound
Cr oss-Rea ctivities. Data were obtained from standard
curves of fenpropathrin, the pyrethroids phenothrin, per-
methrin, resmethrin, fenvalerate, deltamethrin, cypermethrin,
and the metabolites 3-phenoxybenzoic acid and fenpropathrin
acid. In addition, other pesticides such as fenoxycarb, chlor-
propham, and DDT were also tested for cross-reactivity. Each
compound was prepared in 10% methanol in PBS and tested
at the concentration range 0.1-1000 µg/L. The cross-reac-
tivities (CR) were calculated as a ratio of the IC50 of fenpro-
pathrin to that of the tested compound. CR was set at 100%
for fenpropathrin.
RESULTS AND DISCUSSION
Ha p ten Syn th esis a n d Con ju ga tion . The main
steps in the synthesis of haptens are summarized in
Schemes 1 and 2. The intermediate aldehyde 4 was
synthesized by an Ullman coupling (Moroz and Shvarts-
berg, 1974) of the dimethyl acetal of 3-bromobenzalde-
hyde 2 and benzyl 4-hydroxybenzenepropanoate 3 fol-
lowed by acid-catalyzed deprotection in wet solvent. The
synthesis of the analogous tert-butyl ester, prepared by
a more circuitous route as an intermediate for a pyre-
throid radioimmunoassay development, is described by
Demoute and Touer (1987); however, no details of
immunoassay development were given. The intermedi-
ate aldehydes 10 and 11, which lack the terminal phenyl
group of fenpropathrin, were readily prepared by treat-
ment of the potassium salt of 3-hydroxybenzaldehyde
with benzyl bromoacetate and benzyl 6-bromohexanoate
9. Conversion of these aldehydes 4, 10, and 11 to their
cyanohydrins followed by acylation with tetramethyl-
cyclopropanecarbonyl chloride gave the hapten benzyl
esters 5, 12, and 14, respectively. Cleavage of these
benzyl esters with iodotrimethylsilane yielded haptens
6, 13, and 15. Hapten 6 was found to be contaminated
with ∼10% of the tetramethylcyclopropanecarboxylate
ester 6b of 4-hydroxybenzenepropanoic acid, resulting
from the carry-through of unreacted phenol 3 because
of its Rf value being identical to that of the intermediate
aldehyde 4. Identical Rf values for subsequent products
5 and 6a , and 6 and 6b , made removal of these
contaminants extremely difficult. The Rf values of 6 and
6b were identical in a variety of solvents, giving values
ranging from 0.1 to 0.9. Nevertheless, our use of this
preparation of compound 6 as the immunizing hapten
gave excellent antibodies to fenpropathrin. Future
preparation of this material should include a careful
stepwise extraction of the intermediate acetal of com-
pound 4 with aqueous base before proceeding to the
subsequent step. Care should be taken in the extrac-
tion, since any base in excess over the contained phenol
3 can hydrolyze the benzyl ester function. Hapten 8,
substituted by a terminal aromatic amino group, was
synthesized from 3-(4-nitrophenoxy)benzaldehyde (Loewe
and Urbanietz, 1967). Conversion to the cyanohydrin
followed by acylation yielded the nitro-substituted fen-
propathrin 7. Reduction of the nitro group with stan-
nous chloride (Bellamy and Ou, 1984) yielded the amino-
substituted fenpropathrin 8, which was a pale yellow
gum. After TLC development, the product spot rapidly

2218 J. Agric. Food Chem., Vol. 46, No. 6, 1998
Wengatz et al.
Ta ble 4. Deter m in a tion of th e Ra tio of Ha p ten to
P r otein Molecu les F ollow in g Con ju ga tion a
Ta ble 5. Rep r esen ta tive Titer Resu ltsa
coating antigen
hapten molecules
per molecule of
carrier protein
antiserum
8-
13-
15-
conjugate
(MW of carrier protein)
molar ratio
hapten/conjugate
(immunogen) 6-BSA BSA-(B) 13-BSA OVA 15-BSA OVA
6982 (6-KLH) +++
-
nd
+++
nd
nd
++
nd
nd
+++
nd
nd
++
nd
immunogen
56 (8-LPH)
58 (8-THY)
55 (8-fetuin)
nd
nd
nd
+++
++
-
8-fetuin (42 000)
8-thyroglobulin
(660 000)
8-LPH (335 000)
coating antigen
8-BSA (56 000)
5.5 × 10-8/8.8 × 10-10
5.5 × 10-8 /2.5 × 10-10
5.5 × 10-8/5.0 × 10-8
62.5
220
nd
nd
nd
nd
1b
a
Data were determined from a coating antigen concentration
of 1.5 µg/mL and an antibody dilution of 1/80000. All other ELISA
conditions were as described under Materials and Methods.
Antisera and coating antigen combinations showing a titer re-
sponse >0.3 were further screened for inhibition by fenpropathrin.
nd, not determined; -, OD of <0.3; +, OD of 0.3-0.6; ++, OD of
0.6-1.0; +++, OD of 1.0-1.5.
5.5 × 10-8/1.0 × 10-9
55
a
The ratio of hapten to protein molecules was determined
indirectly by the optimized ELISA described under Materials and
Methods. A solution of conjugate with a known protein concentra-
tion was tested for its ability to inhibit the binding of antibodies
in the ELISA. The percent inhibition was then referenced to
equivalent inhibition produced by the hapten alone. The ratio of
this calculated amount of hapten in the conjugate to the concen-
tration of conjugate protein was used to determine the ratios
titers of at least 1/80000 (Table 5) were screened for
competition by fenpropathrin.
b
Scr een in g for Com p et it ion by F en p r op a t h r in .
The results of the competition screening are shown in
Tables 1 and 2. The IC50 values ranged from 20 to 500
µg/L in the homologous and heterologous systems tested.
Concurrently, we were developing immunoassays for
fenvalerate; thus, coating antigens designed for this
assay were tested for binding with fenpropathrin anti-
bodies. Table 1 shows that antibody 6982 was able to
bind to and compete with the fenvalerate antigen.
However, the IC50 for fenpropathrin indicated that
detection limits would be higher than those of conven-
tional methods. Some differences in IC50 were observed
when different coating proteins were used. When the
coating antigen was an OVA conjugate, the IC50 values
were lower compared to those using the BSA conjugates.
The most sensitive assay was a homologous assay that
used antibody 58 and coating antigen 8-BSA. This
assay was used for further assay optimization.
reported above. The LPH conjugate was not completely soluble
during analysis, which may contribute to the low ratio found.
poorly to 8-BSA, and when tested for competition by
fenpropathrin, no competition was seen at the highest
concentrations tested.
Haptens 13 and 15 have aliphatic chains of different
lengths with a terminal carboxy group replacing one
benzyl ring of the fenpropathrin molecule. These hap-
tens were coupled to BSA and OVA by an activated ester
method and used as coating antigens in heterologous
assay systems. Antisera raised against hapten 8 bound
well to these coating antigens, but the IC50 for fenpro-
pathrin was somewhat higher (Table 2) than for the
homologous assay (Table 1). In addition, the length of
the aliphatic chain had no apparent effect on the assay
performance, as the IC50 values were identical.
The number of hapten molecules per molecule of
protein (termed “hapten density”) for the immunogens
was estimated indirectly by competition with the coating
antigen for antibody binding sites (Table 4). The
apparent hapten density varied widely; however, the
antisera that resulted in assays with the greatest
sensitivity were generated from the thyroglobulin con-
jugate, which had the highest hapten density. A higher
hapten density is recommended for the immunogen so
that there are many hapten epitopes available for the
antibody-forming mechanism (Hurn and Chantler, 1980).
Lower hapten densities are desired for the coating
antigens to improve the sensitivity of the ELISA. The
hapten load determined for LPH seemed unreasonably
low and may be due to the difficulty of handling this
protein in solution. Nevertheless, antibodies that could
be used in the ELISA were generated.
An tiser a Ch a r a cter iza tion , Titer . Antiserum ti-
ters that were high enough to begin the competitive
assay screen were obtained after the third immuniza-
tion; maximum titers were obtained after the fifth
immunization. Titers were determined in a checker-
board titration (Gee et al., 1994) in which the concen-
trations of the coating antigen and the antiserum were
varied. All raw antisera showed fairly high titers (up
to 1/100000) to their homologous antigens with the
exception of AS 55, the titer of which was marginal.
Antibodies generated to hapten 6 bound to the homolo-
gous antigen (6-BSA) but did not bind to the heterolo-
gous antigen (8-BSA). Antibodies generated to hapten
8 not only bound the homologous antigen but also bound
well to the heterologous antigens 13-BSA, 13-OVA,
15-BSA, and 15-OVA. Only those systems giving
Assa y Op tim iza tion . Many parameters can influ-
ence the binding of the antibody to the hapten. These
parameters include the pH of the assay buffer during
the competition step and the ionic strength of the buffer.
Organic solvents, gelatin, or detergents are often added
to the assay buffer to improve the solubility of the
analyte or to decrease nonspecific binding. The effects
of these were also determined.
pH Effect. Figure 3 shows the influence of varying
the pH of the assay buffer during the competition step
in the presence of the analyte. Because the control
absorbance varied at each pH, the data are represented
as a percent of the control absorbance at each pH. The
assay sensitivity increased at more acidic or basic pH
values (<5 or >9). However, at basic pH values the
control absorbances decreased. Because the assay was
more sensitive and had higher control absorbances
under more acidic conditions, a pH of 4 was used for
subsequent assays.
Ionic Strength. Because the ionic strength of the
buffer containing the antibody can affect antibody
binding (Tijssen, 1985), the ionic strength of the assay
buffer was varied by increasing the NaCl concentration
from 0.05 to 1.0 M (Figure 4). Increasing the salinity
of the assay buffer resulted in an increase in sensitivity
(IC50 decreased significantly) but also resulted in a
decrease of the control absorbance. The optimum range
of ionic strength was 0.2-0.4 M NaCl.
Solvent Effect. Water-miscible organic solvents were
used to keep fenpropathrin in solution while minimizing
denaturation of the antibody during incubation. Figures
5 shows the effect of methanol, ethanol, acetone, DMF,

Development of a Fenpropathrin ELISA
J. Agric. Food Chem., Vol. 46, No. 6, 1998 2219
F igu r e 2. Structures of compounds tested for cross-reactivity: fenvalerate (16), phenothrin (17), permethrin (18), cypermethrin
(20), resmethrin (19), deltamethrin (21), DDT (23), fenoxycarb (24), chlorpropham (26), 3-phenoxybenzoic acid (22), and
fenpropathrin acid (25).
F igu r e 3. Effect of pH on the sensitivity of the fenpropathrin
F igu r e 4. Effect of the ionic strength of the buffer on the
assay. Assay conditions were as described under Materials and
assay performance. Absorbances are expressed as percent of
Methods using antisera 58 and coating antigen 8-BSA.
the control absorbance to standardize the data sets. The
Absorbance values are corrected for nonspecific binding and
maximum absorbances ranged between 0.835 and 0.244. Data
expressed as percent of the control absorbance. The data points
points are the mean values of duplicates.
are the mean values of duplicates. The maximum absorbances
ranged between 1.04 and 0.98.
ethanol, the absorbance was similar in the presence or
and acetonitrile on the control absorbance. The absor-
bances of the controls containing only organic solvent
increased with increasing concentration of organic
solvent, especially at concentrations >10%. Thus, the
binding of the antibodies to the coating antigen was not
adversely affected and, in fact, was enhanced. An
increase in the absorbance was also observed in the
presence of fenpropathrin. With acetonitrile, DMF, and
absence of fenpropathrin. Since little inhibition was
observed in the presence of fenpropathrin, these sol-
vents were unacceptable for assay purposes. In con-
trast, a concentration of up to 10% methanol or 10%
acetone resulted in high absorbance values in the
absence of fenpropathrin and a significant inhibition of
the signal in the presence of the inhibitor. Thus, 10%
methanol was used in subsequent experiments.

2220 J. Agric. Food Chem., Vol. 46, No. 6, 1998
Wengatz et al.
F igu r e 6. Standard curve for fenpropathrin obtained from a
four-parameter curve fit. The data are corrected for back-
ground and are the averages of three replicates with
coefficient of variation below 15%.
a
for the antibody production. Comparison of homologous
and heterologous assays revealed that the highest
sensitivity was achieved with an homologous assay.
Since heterology often results in more sensitive assays,
the discrepancy may be due to differences in hapten load
among the homologous and heterologous coating anti-
gens. Using antisera 58 in the coating antigen format,
the IC50 was 20 µg/L with a lower detection limit of 2.5
µg/L. This is similar to the detection limit of the GC
method reported by Takimoto et al. (1984)
The assay optimization studies revealed that metha-
nol or acetone at concentrations >10% and assay buffer
at pH 4 resulted in the most sensitive assay. None of
the added detergents or gelatin increased the sensitivity
of the assay. The assay had little cross-reactivity to
other pyrethroids or pyrethroid metabolites, making it
useful for the selective detection of fenpropathrin.
F igu r e 5. Effect of solvents at several concentrations: (A)
with or without 20 µg/L fenpropathrin dissolved in methanol
or ethanol; (B) with and without fenpropathrin dissolved in
DMF, acetone, or acetonitrile. Data points are the mean values
of triplicates.
Effect of Additives. The addition of gelatin has been
found to increase the sensitivity and stability of immu-
noassays (Forlani et al., 1992). Gelatin was tested at
0.1-1.0%. No changes in the assay parameters were
seen except for an increase in the background signal.
A series of nonionic, cationic, and anionic detergents
were added in the competition step to reduce nonspecific
binding and improve solubility of the analyte. At the
concentrations tested, no changes in the fenpropathrin
concentration curve were observed.
The optimized fenpropathrin assay used a 1/8000
dilution of the coating antigen 8-BSA preparation and
antibody 58 at a dilution of 1/40000. This homologous
assay showed an IC50 value of ∼20 µg/L (Figure 6). The
assay detects fenpropathrin within a linear range of
2.5-200 µg/L.
ABBREVIATIONS USED
AS, antiserum; BSA, bovine serum albumin; BSTFA,
bis(trimethylsilyl)trifluoroacetamide; t-BuOH, tert-butyl
alcohol; BuCl, butyl chloride; DAPEC, 1-(3-dimethyl-
aminopropyl)-3-ethylcarbodiimide hydrochloride; DCC,
dicyclohexylcarbodiimide; DMF, dimethylformamide;
DMSO, dimethyl sulfoxide; DMAP, 4-dimethylamino-
pyridine; ELISA, enzyme-linked immunosorbent assay;
GAR/HRP, goat-anti-rabbit immunoglobulin conjugated
to horseradish peroxidase; KLH, hemocyanin of keyhole
limpet; LPH, hemocyanin of Limulus polyphemus; NHS,
N-hydroxysuccinimide; OVA, ovalbumin; PBS, phosphate-
buffered saline; sulfo-NHS, N-hydroxysulfosuccinimide;
THF, tetrahydrofuran; TMSI, iodotrimethylsilane; TMB,
tetramethylbenzidine.
Cr oss-Rea ctivity. The antibodies from antisera 58
were relatively selective for fenpropathrin. Most of the
compounds tested, including a group of pyrethroids, and
pyrethroid metabolites showed no significant cross-
reactivity in the concentration range of 0.1-1000 µg/L
(Table 3), except deltamethrin (CR ) 18.8%).
ACKNOWLEDGMENT
We thank Dr. A. Daniel J ones of the Facility of
Advanced Instrumentation (UCD) for the mass spectral
data and the Department of Chemistry (UCD) for the
use of their NMR instrument.
Con clu sion . The synthesis of different haptens for
fenpropathrin allowed us to test different immunogens

Development of a Fenpropathrin ELISA
J. Agric. Food Chem., Vol. 46, No. 6, 1998 2221
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Received for review December 22, 1997. Revised manuscript
received February 25, 1998. Accepted February 26, 1998. The
research was funded by National Institute of Environmental
Health Sciences Superfund Research Program, 5 P42 ES04699-
08, and Environmental Protection Agency Center for Ecological
Health Research, CR819658, U.S. EPA Cooperative Grant
CR819047, and the USDA Forest Service NAPIAP-R827. UCD
is a NIEHS Health Science Center (1 P30 ESO5707). Al-
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U.S. EPA, no endorsement by the Agency is implied.
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