Synthesis of haptens and protein conjugates for the development of immunoassays for the insect growth regulator fenoxycarb

Article abstract of DOI:10.1021/jf0107050

Sensitive and selective enzyme-linked immunosorbent assays (ELISAs) in the immobilized antigen format were developed for fenoxycarb (1), an insect growth regulator (IGR). The parent molecule [ethyl 2-(4-phenoxyphenoxy)ethylcarbamate] was derivatized at se

Full text of DOI:10.1021/jf0107050

J. Agric. Food Chem. 2002, 50, 29−40 29
Synthesis of Haptens and Protein Conjugates for the
Development of Immunoassays for the Insect Growth Regulator
Fenoxycarb
FERENC SZURDOKI,†,‡ ANDRA´ S SZE´KA´ CS,§ HONG M. LE,§ AND
BRUCE D. HAMMOCK*,†
Department of Entomology and Cancer Research Center, University of California,
1 Shields Avenue, Davis, California 95616, and Plant Protection Institute,
Hungarian Academy of Sciences, H-1525 Budapest, POB 102, Hungary
Sensitive and selective enzyme-linked immunosorbent assays (ELISAs) in the immobilized antigen
format were developed for fenoxycarb (1), an insect growth regulator (IGR). The parent molecule
[ethyl 2-(4-phenoxyphenoxy)ethylcarbamate] was derivatized at several positions to obtain haptens
(2-5) that were used to produce protein conjugates and rabbit polyclonal antisera. Amino derivatives
of fenoxycarb at the terminal and internal rings (2 and 3, respectively) were linked to carrier proteins
by azo coupling. Carboxyalkyl-spacer groups were attached to the ethyl group and the nitrogen atom
of the target compound (1) to obtain haptens 4 and 5, respectively. Hapten-homologous ELISAs
based on protein conjugates of compounds 2 and 4 determined fenoxycarb in the mid-ppb range
(IC50, 102 and 95 ppb, respectively). A more sensitive hapten-heterologous ELISA (IC50, 17 ppb;
detection limit 0.5 ppb) involved the antiserum raised against a conjugate of hapten 2 and the plate-
coating antigen obtained from compound 3. These assays displayed no significant interferences with
photodegradation products of fenoxycarb, the IGRs methoprene and pyriproxyfen, and a variety of
pesticides including the pyrethroids fenvalerate and cypermethryn, the phenoxyacetic acid herbicide
2,4-D, DDT, and the nitrodiphenyl ether herbicides acifluorfen and fluorodifen.
KEYWORDS: Fenoxycarb; hapten design; synthesis of haptens and protein conjugates; polyclonal
antibodies; ELISA; immunoassay; heterology
INTRODUCTION
Environmental contamination from pesticides is an increasing
concern for the public and regulatory agencies. Routine
monitoring of these pollutants is required because of their human
and environmental health hazards. Rapid, field-portable, and
inexpensive methods of analysis also assist with product
stewardship by facilitating timely and effective use of the
pesticides. Immunoassays provide a simple and economical
alternative to instrumental methods for environmental and
agricultural trace analyses (1-4). As part of a collaborative
research and development program (1, 3, 5), enzyme-linked
immunosorbent assays (ELISAs) were developed for fenoxy-
carb, a novel insect growth regulator (IGR) insecticide (1, Figure
1).
Figure 1. Structure of fenoxycarb (1).
a wide range of insects (6-10). Recent studies shed light on
important details of the mode of action of fenoxycarb (11-
14). This nonneurotoxic carbamate (1) is used for insect control
in agriculture, forestry, and stored products, and is also employed
as a public health insecticide (6, 8, 15-17). Fenoxycarb is
registered for control of caterpillars, psyllids, and scales in
Europe, and for fire ants in the U.S. (18, 19).
Like most IGRs, fenoxycarb has remarkably low mammalian
toxicity and is much more insect-selective than the conventional
insecticides (15-17, 20). Fenoxycarb is nontoxic to a number
of beneficial insects and can be included in integrated pest
management systems (6, 16, 21-23). However, this IGR (1) is
harmful to some species of aquatic invertebrates, fish, and
nontarget insects (8, 16, 18, 19, 24-26). The silkworm, Bombyx
mori, is particularly sensitive to fenoxycarb (8, 27). Thus, this
pesticide (1) has recently been suspected to cause the nonspin-
Fenoxycarb (Ro 13-5223, Insegar, 1) exhibits potent insect
juvenile hormone-mimic activity, and thus causes serious
disturbances in the development, reproduction, and behavior of
* To whom correspondence should be addressed. Phone: 530-752-7519.
Fax: 530-752-1537. E-mail: bdhammock@ucdavis.edu.
† University of California.
‡ Current address: Polestar Technologies, Inc., 220 Reservoir Street, Suite
32, Needham Heights, MA 02494.
§ Hungarian Academy of Sciences.
10.1021/jf0107050 CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/22/2001

30 J. Agric. Food Chem., Vol. 50, No. 1, 2002
Szurdoki et al.
Figure 2. Syntheses leading to hapten 2 and conjugates 14 (Prot, carrier protein).
(6) were available from our related study (10) and synthesized according
to known procedures (34). Protein concentrations of the conjugates were
determined by the BCA protein assay (Pierce, Rockford, IL) following
the manufacturer’s instructions.
ning syndrome resulting in considerable economical losses in
silkworm breeding (8, 14, 28).
Chromatographical techniques to detect residues of this
pesticide involve laborious extraction and cleanup before
analysis as well as costly instrumentation (15, 19, 29-32).
Therefore, immunoassays with no or minimal sample pretreat-
ment would facilitate the inexpensive and high-sample-
throughput determination of fenoxycarb in environmental and
agricultural samples. Recently, Giraudi et al. (28) published their
results with a fenoxycarb ELISA based on a hapten comprising
a part of the target structure (1). In our earlier studies, screening
of several anti-fenoxycarb antisera led to preliminary ELISAs
for this IGR (1) (3, 33). This paper focuses on the synthesis of
a panel of haptens (2-5, Figures 2-5) used to develop our
antisera and immunoassays for fenoxycarb, and on the charac-
terization of three resulting ELISAs.
Instruments. Microtiter plates were read using an iEMS electronic
microplate reader (Labsystems, Helsinki, Finland) controlled by the
software Ascent provided by the vendor of the reader. Melting points
(uncorrected) were taken with a Thomas-Hoover capillary apparatus.
Ultraviolet-visible (UV-Vis) spectra were recorded on a Jasco 7850
spectrometer (Jasco Corp., Tokyo, Japan) and a Shimadzu UV-1601
spectrophotometer (Shimadzu Corp., Kyoto, Japan); wavelengths of
characteristic peaks (λmax, nm) are reported. Molar extinction coefficient
(ꢀ, L/mol/cm) values, given in parentheses, were also determined for
certain compounds. Infrared (IR) spectra were recorded on a Perkin-
Elmer 1600 series Fourier transform IR spectrometer; wavenumber
values (λmax, 1/cm) are given. NMR spectra were obtained with a
General Electric QE-300 spectrometer (Bruker NMR, Billerica, MA)
operating at 300 MHz for 1H- and 75 MHz for 13C-nuclei, if not
1
otherwise stated. Some H NMR spectra were obtained on a Bruker
MATERIALS AND METHODS
AW-80 (Bruker, Karlsruhe, Germany) instrument operating at 80 MHz.
Chemical shifts (δ, ppm) are given relative to tetramethylsilane as
internal reference. Electron impact mass spectra (MS) were determined
on two instruments (A and B); data are reported as m/z, relative
intensities (%) are given in parentheses. Instrument A consisted of the
following: Trio-2 (VG Masslab, Altrincham, U.K.) apparatus using
70 eV electron ionization. Gas chromatography (GC) separations for
Reagents. Common reagents and organic solvents were purchased
from Aldrich Chemical Co. (Milwaukee, WI), and immunochemicals
were obtained from Sigma Chemical Co. (St. Louis, MO) unless
otherwise indicated. Aqueous solutions used in immunoassays and
experiments with protein conjugates were prepared with Nanopure
purified water. Fenoxycarb (1) and 2-(4-phenoxyphenoxy)ethylamine

Development of ELISAs for IGR Fenoxycarb
J. Agric. Food Chem., Vol. 50, No. 1, 2002 31
washings were concentrated under reduced pressure to give 0.64 g (92%
based on 96% purity of the product) of oily ester 8. IR (NaCl) 3340,
1
1696, 1265. H NMR (CDCl3) 1.24 (t, J ) 7 Hz, 3 H), 3.15 (b, 1 H),
3.33 (m, 2 H), 3.69 (t, J ) 5 Hz, 2 H), 4.12 (q, J ) 7 Hz, 2 H), 5.38
(b, 1 H). 13C NMR (CDCl3) 14.24, 43.04, 60.73, 61.40, 157.32. GC-
MS (instrument B, program C) main peak (5.5 min, 96%) 88 (33), 102
(100), 133 (4, M+). The reported NMR spectra show the signals of
compound 8, the major component. This product was used without
further purification in the following reaction.
Ethyl 2-(4-Methylphenylsulfonyloxy)ethylcarbamate (9). The
experiment was carried out with stirring under nitrogen. p-Toluene-
sulfonyl chloride (98%, 0.856 g, 4.4 mmol) was added to a solution of
the alcohol 8 (96%, 0.555 g, 4.0 mmol) and 4-(dimethylamino)pyridine
(24.4 mg, 0.2 mmol) in anhydrous pyridine (5 mL) at 4 °C. The reaction
mixture was stirred at 4 °C for 30 min, and at room-temperature
overnight, then diluted with hexane/ethyl acetate 2:1 mixture (20 mL),
then acidified by dropwise addition of 6 M HCl solution at 4 °C. The
organic phase was separated, and the aqueous phase was extracted with
hexane/ethyl acetate 2:1 mixture (4 × 5 mL). The combined organic
extracts were washed with water (2 × 5 mL), saturated NaHCO3
solution (2 × 5 mL), and water (3 × 5 mL) again. The solution was
dried over Na2SO4, then concentrated under reduced pressure. The
product was purified by recrystallyzation from hexane/ethyl acetate to
yield 0.7 g (61%) of the solid tosilate 9. mp 77-80 °C. IR (KBr) 3307,
1
1680, 1361, 1272, 1180, 814. H NMR (CDCl3) 1.21 (t, J ) 7 Hz, 3
H), 2.45 (s, 3 H), 3.43 (m, 2 H), 4.0-4.2 (m, 4 H), 4.98 (b, 1 H), 7.36
(d, J ) 8 Hz, 2 H), 7.79 (d, J ) 8 Hz, 2 H). 13C NMR (CDCl3) 14.31,
21.36, 39.75, 60.72, 68.95, 127.98, 129.73, 132.28, 144.83, 156.30.
4-(4-Nitrophenoxy)phenol (12). The experiment was carried out
using anhydrous reagents and solvent with vigorous stirring under
nitrogen. 1-Fluoro-4-nitrobenzene (10, 99%, 2.14 mL, 20 mmol) was
added dropwise to the mixture of hydroquinone (11, 99%, 2.45 g, 22
mmol), finely ground K2CO3 (13.8 g, 100 mmol), and N,N-dimethyl-
formamide (DMF, 25 mL). The reaction mixture was heated to its
boiling point in 11/2 h, kept at reflux temperature overnight, cooled to
ambient temperature, then slowly poured into a stirred mixture of 6 M
HCl (40 mL) and crushed ice (100 g). The resulting precipitate was
collected by filtration, washed thoroughly with water, then dried in a
vacuum-desiccator over KOH to yield 4.5 g of crude product. A portion
(0.7 g) of this solid material was purified by preparative TLC using a
solvent of hexane/ethyl acetate (3:2) to yield 0.4 g (corresponds to 56%
overall yield) of 12 as a yellow crystalline product. mp 166-169 °C
[lit. 171-172 °C (36)]. IR (KBr) 3436, 1589, 1505, 1342, 1240, 845.
1H NMR (CDCl3/d6-DMSO) 6.8-7.15 (m, 6 H), 8.16 (d, J ) 9 Hz, 2
H), 9.24 (b, 1 H). 13C NMR (CDCl3/d6-DMSO) 116.00, 116.61, 121.60,
125.67, 141.63, 146.20, 154.69, 164.16. GC-MS (instrument B,
program C) main peak (13.8 min) 173 (9), 185 (3), 201 (5), 215 (8),
231 (100, M+).
Figure 3. Syntheses leading to hapten 3 and conjugates 16 and 16′
(Prot, carrier protein).
GC-MS analyses were done by a Hewlett-Packard 5890 instrument
equipped with a DB-5 column (15 m × 0.25 mm i.d.; film thickness,
0.25 µm) interfaced to this mass spectrometer. Helium was employed
as carrier gas at a velocity of 35 cm/sec. Split injections with a split
ratio of 25:1 were used. The injector temperature was 250 °C, the ion
source temperature was 200 °C, and the transfer line was held at 300
°C. Instrument B was the following: Varian Saturn 4D mass spec-
trometer (Varian Assoc., Walnut Creek, CA). A Varian 3400 CX gas
chromatograph equipped with a DB-5 column (30 m × 0.25 mm i.d.)
was interfaced to this mass spectrometer for GC-MS analyses, and
was operated in splitless mode. Further parameters were similar to those
reported for instrument A. GC separations were performed with column
temperature programs as follows: Program A was 160 °C for 2 min,
160 to 300 °C at 10 °C/min, and 300 °C for 10 min. Program B was
50 °C for 1 min, 50 to 300 °C at 15 °C/min, and 300 °C for 10 min.
Program C was 40 °C for 1 min, 40 to 300 °C at 20 °C/min, and 300
°C for 5 min. For GC-MS separations, retention time (min) and relative
peak intensity (%) are given in parentheses. Electrospray mass spectra
in positive mode (MS-ES+) were recorded by a VG Quatro-BQ triple
quadrupole mass spectrometer (VG Biotech, Altrincham, U.K.); samples
were introduced in a mixture of acetonitrile/water (1:1). Data are
reported as m/z. Elemental analysis data were determined by the
combustion method.
Ethyl 2-[4-(4-Nitrophenoxy)phenoxy]ethylcarbamate (13). The
experiment was performed using anhydrous reagents and solvent with
stirring under nitrogen. A mixture of the phenol 12 (243 mg, 1.05
mmol), the tosilate 9 (287 mg, 1.0 mmol), finely ground K2CO3 (691
mg, 5.0 mmol), and DMF (10 mL) was kept at 50 °C overnight. The
reaction mixture was poured into a stirred mixture of ether (20 mL)
and ice-water (40 mL). The organic layer was separated, and the
aqueous layer was extracted by ether (2 × 5 mL). The combined Etheral
solutions were successively washed with saturated NaHCO3 solution
(10 mL) and water (4 × 5 mL), dried over Na2SO4, and evaporated
under reduced pressure. The residue was purified by preparative TLC
using a solvent of hexane/ethyl acetate (3:2) to yield 203 mg (59%) of
13 as an oily product. IR (NaCl) 3339, 1709, 1590, 1508, 1342, 1231,
Synthetic Procedures. Syntheses of haptens and protein conjugates
are outlined in Figures 2-5. Yields of hapten synthesis steps were
moderate to good. Spectral data consistent with the structures are
provided.
Safety Notice. During preparation of compound 15, the temperature
of the nitration reaction mixture must be kept at or below room
temperature because of the explosive nature of acetyl nitrate (35). A
protective shield must be used, and special care must be exercised
throughout this operation.
1
845. H NMR (CDCl3) 1.26 (t, J ) 7 Hz, 3 H), 3.4-3.7 (m, 2 H),
Ethyl 2-Hydroxyethylcarbamate (8). The experiment was carried
out using anhydrous reagents and solvent with vigorous stirring under
nitrogen. Ethanolamine (7, 99%, 305 µL, 5.0 mmol) was added to a
mixture of tetrahydrofuran (THF, 15 mL), finely ground potassium
fluoride (1.45 g, 25 mmol), and potassium carbonate (3.46 g, 25 mmol).
Ethyl chloformate (97%, 517 µL, 5.25 mmol) was added dropwise with
ice-cooling in 10 min; the reaction mixture was maintained at room
temperature for 1 h, then at reflux temperature overnight. The mixture
was cooled to room temperature and filtered. The solid material on the
filter was washed with ether (3 × 10 mL). The combined filtrate and
4.0-4.3 (m, 4 H), 5.16 (b, 1 H), 6.85-7.15 (m, 6 H), 8.18 (d, J ) 9
Hz, 2 H). MS (instrument A) 88 (100), 116 (100), 139 (28), 231 (40),
300 (26), 346 (5, M+).
Ethyl 2-[4-(4-Aminophenoxy)phenoxy]ethylcarbamate (2). Ni-
trogen atmosphere was maintained during the experiment. Palladium
on activated carbon (palladium content, 10%; 61 mg) was suspended
in water (2 mL) before use to avoid the fire hazard that would be caused
by methanol being in contact with a dry catalyst in the presence of air
(37). A solution of the nitro compound 13 (122 mg, 0.352 mmol) in

32 J. Agric. Food Chem., Vol. 50, No. 1, 2002
Szurdoki et al.
Figure 4. Syntheses leading to hapten 4 and conjugates 20, 20′, and 20′′ (Prot, carrier protein).
Figure 5. Syntheses leading to hapten 5 and conjugates 23 (Prot, carrier protein).
methanol (15 mL), and then ammonium formate (97%, 229 mg, 3.52
mmol), were added to the wetted catalyst with stirring at 4 °C. The
reaction mixture was stirred at room temperature overnight, and the
catalyst was filtered off and washed with methanol (2 × 7.5 mL) under
nitrogen. The combined filtrate and washings were concentrated under
diminished pressure. Ether (10 mL) and 5% NaHCO3 solution (25 mL)
were then added with stirring. The organic layer was separated, and
the aqueous layer was extracted with ether (2 × 7.5 mL). The combined
organic layers were washed with water (5 × 5 mL), dried over Na2-
SO4, and evaporated under reduced pressure. The residue was purified
by preparative TLC using a solvent of hexane/ethyl acetate (1:2) to
yield 80 mg (72%) of 2 as an oily product. An analytical sample was
obtained by crystallyzation from carbon tetrachloride. mp 78-79 °C.
IR (KBr) 3436, 3313, 1672, 1541, 1498, 1214, 829. 1H NMR (CDCl3)
1.24 (t, J ) 7 Hz, 3 H), 3.56 (m, 2 H), 3.75 (b, 2 H), 3.98 (t, J ) 5 Hz,
2 H), 4.12 (q, J ) 7 Hz, 2 H), 5.13 (b, 1 H), 6.65 (d, J ) 9 Hz, 2 H),
6.7-7.15 (m, 6 H). 13C NMR (CDCl3) 14.48, 40.35, 60.82, 67.30,
115.23, 116.06, 118.85, 119.90, 142.05, 149.63, 152.30, 153.70, 156.62.
GC-MS (instrument A, program B) main peak (16.7 min) 88 (56), 92
(16), 116 (47), 201 (100), 270 (49), 316 (19, M+).
Protein Conjugates of Hapten 2 (14). Hapten 2 (7.9 mg, 25 µmol)
was dissolved in a mixture of DMF (2 mL) and aqueous HCl (0.5 M,
0.5 mL). The diazotation reaction was then carried out with stirring at
4 °C. An aqueous solution of sodium nitrite (0.1 M, 0.3 mL) was added
dropwise to the hapten solution. After 30 min, urea (0.9 mg, 15 µmol)
was added to decompose excess nitrous acid. After a further 5 min,
the reaction mixture was divided into four equal portions. Meanwhile,
each carrier protein was dissolved in 0.15 M Na2B4O7‚10 H2O (Borax)
solution (5.5 mL) with stirring, and the solutions were then cooled to
4 °C. The proteins (15 mg each) used were bovine serum albumin
(BSA), keyhole limpet hemocyanin (KLH), ovalbumin (OVA), and
porcine thyroglobulin (TYG). Each aliquot of the diazonium salt
solution was then added dropwise to a rapidly stirred protein solution
at 4 °C. The reaction mixtures were stirred at 4 °C overnight, and at
room temperature for 2 h, then the resulting yellow conjugates were
purified by exhaustive dialysis in 0.01 M phosphate-buffered saline

Development of ELISAs for IGR Fenoxycarb
J. Agric. Food Chem., Vol. 50, No. 1, 2002 33
(0.8% NaCl), pH 7.5 (PBS). The conjugates of proteins BSA, KLH,
OVA, and TYG were designated as 14-BSA, 14-KLH, 14-OVA, and
14-TYG, respectively; UV-Vis (PBS) 355-378.
in PBS. The conjugates of proteins BSA, CONA, LPH, OVA, and TYG
were designated as 16-BSA, 16-CONA, 16-LPH, 16-OVA, and 16-
TYG, respectively. UV-Vis (PBS) 392-396.
Modified Procedure to Obtain Protein Conjugates (16′) of
Hapten 3 using the Anhydrous Diazotation Method. The diazotation
reaction was carried out with stirring under nitrogen using dry DMSO
in a vial cooled in a water bath kept at about 19-20 °C. Care was
exercised to avoid solidification of the reaction mixture because the
melting point of DMSO is 18 °C. Hapten 3 (28.5 mg, 90 µmol) was
dissolved in a solution of sulfuric acid in DMSO (0.05 M, 2.25 mL,
112.5 µmol). A solution of butyl nitrite in DMSO (0.05 M, 2.25 mL,
112.5 µmol) was added dropwise to this solution. After 10 min, the
resulting diazonium salt solution was divided into six equal portions.
Each aliquot was added dropwise to a vigorously stirred solution of a
carrier protein (15 mg of BSA, CONA, KLH, LPH, OVA, or TYG) in
Borax solution (0.1 M, 7.5 mL) at 4 °C. The reaction mixtures were
stirred at 4 °C for 11/2 h, and then stirred at room temperature overnight.
The yellow conjugates were purified by exhaustive dialysis in PBS.
The conjugates of proteins BSA, CONA, KLH, LPH, OVA, and TYG
were designated as 16′-BSA, 16′-CONA, 16′-KLH, 16′-LPH, 16′-
OVA, and 16′-TYG, respectively. UV-Vis (PBS) 395-399.
Ethyl 2-(4-Phenoxy-2/3-nitrophenoxy)ethylcarbamate (15). The
experiment was carried out with intensive stirring under nitrogen. Acetyl
chloride (98%, 120 µL, 1.65 mmol) was added dropwise to the solution
of fenoxycarb (1, 0.452 g, 1.5 mmol) and silver nitrate (0.280 g, 1.65
mmol) in dry acetonitrile (5 mL) at 4 °C. The resulting suspension
was kept at the same temperature for 30 min, then at room temperature
for 11/2 h. Care has to be exercised that the temperature of the reaction
mixture is kept at or below room temperature throughout the operation
to avoid explosion (see Safety Notice above). The reaction mixture
was poured into a stirred mixture of saturated NaCl solution (20 mL)
and chloroform (10 mL) at 4 °C. The mixture was filtered, and the
precipitate on the filter was washed with saturated NaCl solution (2 ×
2.5 mL) and chloroform (2 × 5 mL). The chloroform-phase of the
combined filtrates and washings was separated, and the aqueous phase
was extracted with chloroform (3 × 3 mL). The combined chloroform
solutions were successively washed with saturated NaHCO3 (2 × 5
mL) and water (4 × 5 mL), dried over Na2SO4, and then concentrated
under reduced pressure. The crude product was purified by preparative
TLC using a solvent of hexane/ethyl acetate (1:1) to yield 351 mg (68%)
of 15 as an oily product. IR (NaCl) 3336, 1712, 1589, 1528, 1487,
1350, 1268, 1216. 1H NMR (CDCl3) 1.24 (t, J ) 7 Hz, 3 H), 3.61 (m,
2 H), 4.0-4.3 (m, 4 H), 5.35 (b, 1 H), 6.85-7.55 (m, 8 H). 13C NMR
(CDCl3) 14.45, 40.11, 60.94, 69.43, 115.74, 116.28, 118.63, 123.98,
124.41, 124.76, 129.79, 129.98, 139.80, 147.81, 150.48, 156.46, 156.66.
GC-MS (instrument A, program A) major isomer (9.5 min, ca. 94%)
77 (25), 88 (80), 116 (100), 231 (10), 300 (4), 346 (0.5, M+); minor
isomer (9.8 min, ca. 6%) 77 (18), 88 (100), 116 (82), 231 (3), 300 (2),
346 (0.5, M+). The NMR spectra represent the signals of the major
isomer. No efforts were made to identify or isolate the two isomers.
Ethyl 2-(4-Phenoxy-2/3-aminophenoxy)ethylcarbamate (3). The
experiment was carried out with stirring under nitrogen. Palladium on
activated carbon (palladium content, 10%; 200 mg) was suspended in
water (4 mL); a solution of the nitro compound 15 (242 mg, 0.7 mmol)
in methanol (30 mL), and then ammonium formate (97%, 455 mg, 7.0
mmol) were added to the suspension at 4 °C. The reaction mixture
was stirred at room-temperature overnight, and then the catalyst was
filtered off and washed with methanol (3 × 5 mL) under nitrogen.
The combined filtrate and washings were concentrated under diminished
pressure. Ethyl acetate (20 mL) and saturated NaHCO3 solution (10
mL) were then added with stirring. The organic layer was separated,
and the aqueous layer was extracted with ethyl acetate (3 × 3 mL).
The combined organic layers were washed with saturated NaCl solution
(4 × 5 mL), dried over Na2SO4, and evaporated under reduced pressure.
The residue was purified by preparative TLC using a solvent of hexane/
ethyl acetate (1:1) to yield 151 mg (68%) of 3 as a solid product. mp
5-Ethoxycarbonylpentyl 2-(4-Phenoxyphenoxy)ethylcarbamate
(19). The experiment was carried out with stirring under nitrogen. A
solution of ethyl 6-hydroxyhexanoate (17, 97%, 1.16 g, 7.0 mmol) and
4-nitrophenyl chloroformate (18, 97%, 1.455 g, 7.0 mmol) in anhydrous
pyridine (30 mL) was allowed to react at room temperature overnight.
2-(4-Phenoxyphenoxy)ethylamine (6, 1.605 g, 7.0 mmol) was then
added, and the reaction mixture was kept at 55-60 °C for 24 h. The
solution was diluted with water (160 mL) and extracted with dichlo-
romethane (3 × 150 mL). The collected organic extracts were
successively washed with 10% HCl (3 × 150 mL), saturated Na2CO3
(3 × 150 mL), and saturated NaCl solutions (150 mL), dried over Na2-
SO4, and evaporated under diminished pressure. The resulting crude
product was purified by column chromatography using a solvent of
chloroform/ethyl acetate (9:1) to provide 2.35 g (81%) of 19 as a solid
product. mp 47 °C. Anal. found: C, 66.4, H, 7.2%. Calcd. for C23H29-
NO6: C, 66.49; H, 7.03%. IR (KBr) 3316, 1731, 1687, 1506, 1490,
1
1466, 1222, 1196. H NMR (CDCl3, 80 MHz) 1.18 (t, J ) 7.5 Hz,
3H), 1.3-1.85 (m, 6H), 2.23 (t, J ) 7 Hz, 2H), 3.49 (m, 2H), 3.9-
4.25 (m, 6H), 5.0 (b, 1H), 6.7-7.05 (m, 7H), 7.05-7.35 (m, 2H). MS-
ES+ 416 (M + H+).
5-Carboxypentyl 2-(4-Phenoxyphenoxy)ethylcarbamate (4). A
solution of ester 19 (1.04 g, 2.5 mmol) in 35 mL of ethanol was added
to 1 M NaOH (3.0 mL), and the mixture was stirred at reflux
temperature for 8 h. The reaction mixture was then concentrated under
reduced pressure, diluted with water (100 mL), and cooled in an ice
bath. After addition of ether (100 mL), the stirred mixture was acidified
with 1 M HCl. The aqueous phase was separated, then extracted with
ether (4 × 50 mL). The combined organic solutions were dried over
Na2SO4 and concentrated under diminished pressure. The precipitated
crystalline product was collected by filtration to afford 0.89 g (92%)
of 4. mp 114-115 °C. Anal. found: C, 65.2, H, 6.4%. Calcd. for C21H25-
NO6: C, 65.10; H, 6.50%. IR (KBr) 3500-2500, 1696, 1508, 1490,
1468, 1241, 1210. 1H NMR (CDCl3, 80 MHz) 1.3-1.85 (m, 6H), 2.27
(t, J ) 7 Hz, 2H), 3.50 (m, 2H), 3.85-4.2 (m, 4H), 5.08 (b, 1H), 6.7-
7.05 (m, 7H), 7.1-7.35 (m, 2H), 9.24 (b, 1H). MS-ES+ 388 (M +
H+).
Protein Conjugates of Hapten 4 (20, 20′, and 20′′). Compound 4
(20 mg, 51.6 µmol) and N-hydroxysuccinimide (NHS, 6.9 mg, 60 µmol)
were dissolved in dry THF (1 mL), and dicyclohexylcarbodiimide
(DCC, 12.4 mg, 60 µmol) was added to this solution. The reaction
mixture was stirred at room temperature for 2 h, and the precipitated
solid (dicyclohexylurea) was removed by filtration. The resulting active
ester solution was divided into two aliquots (300 µL and 700 µL). Each
aliquot was added dropwise to a solution of BSA (100 mg) in water
(10.5 mL) and THF (0.6 mL) with stirring at 4 °C (reagent ratios, 0.16
and 0.36 µmol hapten/mg protein, respectively). The reaction mixtures
were stirred for 24 h at 4 °C. The conjugates were then purified by
exhaustive dialysis in water. Employing a similar method, hapten 4
was also conjugated to BSA, CONA, and KLH using a 1 µmol hapten/
mg protein reagent ratio. The conjugates of proteins BSA (reagent
1
83-86 °C. IR (KBr) 3409, 3313, 1708, 1594, 1511, 1268, 1222. H
NMR (CDCl3) 1.23 (t, J ) 7 Hz, 3 H), 3.57 (m, 2 H), 3.88 (b, 2 H),
4.02 (t, J ) 5 Hz, 2 H), 4.12 (q, J ) 7 Hz, 2 H), 5.28 (b, 1 H), 6.3-
7.4 (m, 8 H). 13C NMR (CDCl3) 14.60, 40.48, 60.87, 68.06, 106.81,
108.57, 112.67, 117.93, 122.19, 122.50, 129.27, 129.56, 137.54, 142.27,
151.08, 156.73, 158.12. GC-MS (instrument A, program A) major
isomer (9.4 min, ca. 98%) 77 (19), 88 (100), 116 (83), 200 (52), 201
(27), 270 (13), 316 (6, M+); minor isomer (9.8 min, ca. 2%) 77 (22),
88 (100), 116 (64), 200 (43), 201 (29), 270 (14), 316 (4, M+). The
NMR spectra represent the signals of the major isomer. No efforts were
made to identify or isolate the two isomers.
Protein Conjugates of Hapten 3 (16). Hapten 3 (31.6 mg, 100
µmol) was dissolved in aqueous HCl (0.5 M, 1.0 mL). The diazotation
reaction was then carried out with stirring at 4 °C. An aqueous solution
of sodium nitrite (0.2 M, 0.55 mL, 110 µmol) was added dropwise to
the hapten solution. After 30 min, urea (1.8 mg, 30 µmol) was added.
After a further 5 min, the reaction mixture was divided into five equal
portions. Each aliquot was then added dropwise to a rapidly stirred
solution of a carrier protein (20 mg) in Borax solution (0.2 M, 5 mL)
at 4 °C. The proteins used were BSA, conalbumin (CONA), hemocyanin
of Limulus polyphemus (LPH), OVA, and TYG. The reaction mixtures
were stirred at 4 °C overnight and at room temperature for 2 h, and
then the resulting yellow conjugates were purified by exhaustive dialysis

34 J. Agric. Food Chem., Vol. 50, No. 1, 2002
Szurdoki et al.
ratios: 0.16, 0.36, and 1 µmol hapten/mg protein), CONA, and KLH
were designated as 20-BSA, 20′-BSA, 20′′-BSA, 20′′-CONA, and 20′′-
KLH, respectively; UV-Vis (PBS) 290-310. Lacking significant peaks
above 310 nm, the UV-Vis spectra of conjugates 20, 20′, and 20′′
were not useful for the determination of hapten/protein ratios because
the hapten and protein bands overlapped.
Ethyl N-[2-(4-Phenoxyphenoxy)ethyl]-N-(5-ethoxycarbonylpen-
tyl)-carbamate (22). The procedure was similar to that of reference
53. The experiment was carried out using fenoxycarb (1, 0.452 g, 1.5
mmol), a suspension of NaH (60% dispersion in mineral oil, 72 mg,
1.8 mmol), and ethyl 6-bromohexanoate (99%, 324 µL, 1.8 mmol).
The crude product was purified by preparative TLC using a solvent of
hexane/ethyl acetate (3:2) to yield 0.56 g (84%) of 22 as an oily product.
1H NMR: (CDCl3) 1.25 (t, J ) 7 Hz, 3 H), 1.26 (t, J ) 7 Hz, 3 H),
1.3-2.0 (m, 6 H), 2.30 (t, J ) 7.5 Hz, 2 H), 3.34 (m, 2 H), 3.58 (t, J
) 5 Hz, 2 H), 3.95-4.25 (m, 6 H), 6.8-7.1 (m, 7 H), 7.2-7.35 (m, 2
H).
°C. UV-Vis (methanol) 324 (ꢀ ) 19,100). IR (KBr) 1585, 1489, 1477,
1
1254, 1208, 790, 691. H NMR (CDCl3,) 2.3 (s, 3 H), 6.92 (d, 1 H),
7.0-7.2 (m, 5 H), 7.3-7.5 (m, 4 H), 7.60 (d, 1 H), 7.68 (s, 1 H), 12.5
(s, OH).
4-Methyl-2-(4-phenoxyphenyl)azo-phenol (26c). This compound
was also synthesized in a similar way from 4-phenoxyaniline (24c).
251 mg (82%) of 26c as a crystalline product was isolated. mp 62-63
°C. UV-Vis (methanol) 342 (ꢀ ) 20,900). IR (KBr) 1583, 1488, 1435,
1278, 1240, 849. 1H NMR (CDCl3) 2.3 (s, 3 H), 6.90 (d, 1 H), 6.98-
7.18 (m, 6 H), 7.3-7.5 (m, 2 H), 7.7 (s, 1 H), 7.84 (d, 2 H), 12.6 (s,
OH).
Immunization and Antiserum Preparation. A routine immuniza-
tion protocol (1) was followed. Conjugates 14-KLH, 14-TYG, 16-
LPH, 16-TYG, 20′′-CONA, 20′′-KLH, and 23-KLH were used as
immunogens. Rabbit polyclonal antisera were collected as described
by Sze´ka´cs and Hammock (1).
ELISA. ELISA experiments in the immobilized antigen format were
performed following a modified version of a protocol described earlier
(1). 96-Well microtiter plates (Nunc, Roskilde, DK, #442404) were
coated with appropriate fenoxycarb-protein conjugates which had been
dissolved in carbonate buffer (0.1 M, pH 9.6), and then blocked with
a 1% solution of gelatin (Reanal, Hungary) in PBS. Sample or standard
solution and the antiserum solution, both in PBS containing 0.05%
Tween 20 (PBST), were dispensed into the wells sequentially. After
incubation, the wells were treated with goat anti-rabbit IgG (H + L)-
horseradish peroxidase conjugate (Bio-Rad Laboratories, Richmond,
CA, 1:12,000 dilution in PBST). Enzymatic activity was then measured
using a chromogenic substrate solution containing o-phenylenediamine
and hydrogen peroxide (1). The optical density (OD) values at 492 nm
were recorded. To construct standard curves, stock solutions of
fenoxycarb or related compounds in methanol or acetonitrile (10 mg/
mL) were serially diluted with PBST. Sigmoidal standard curves were
generated from the measured OD values using the common four-
parameter curve fit method (1). The IC50 value is the analyte
concentration required for 50% inhibition. The lower detection limit
(LDL) is defined as the analyte concentration reducing the mean blank
OD value by three standard deviations (SD) of the blank reading.
Ethyl N-[2-(4-phenoxyphenoxy)ethyl]-N-(5-carboxypentyl)-car-
bamate (5). The experimental method was similar to that employed to
prepare compound 4 (above). A solution of ester 22 (490 mg, 1.105
mmol) in ethanol (20 mL) and 1 M NaOH (1.66 mL) was used. The
crude product was purified by preparative TLC using a solvent of
hexane/ethyl acetate/acetic acid (15:15:1) to give 0.24 g (52%) of 5 as
an oily substance. UV-Vis (THF) 271, 279. IR (NaCl) 3500-2500,
1705, 1656, 1615, 1439, 1198. 1H NMR (CDCl3) 1.27 (t, J ) 7 Hz, 3
H), 1.3-2.0 (m, 6 H), 2.35 (t, J ) 7.5 Hz, 2 H), 3.34 (m, 2 H), 3.61
(m, 2 H), 3.95-4.25 (m, 4 H), 6.8-7.1 (m, 7 H), 7.2-7.35 (m, 2 H),
9.87 (b, 1 H). 13C NMR (CDCl3) 14.46, 24.22, 25.96, 28.04, 33.70,
47.11, 48.21, 61.27, 66.65, 115.23, 117.40, 120.59, 122.27, 129.40,
150.12, 154.65, 156.48, 158.19, 180.6. MS 77 (16), 184 (57), 186 (10),
199 (1.3), 230 (100), 342 (0.16), 370 (0.33), 398 (0.12) 415 (0.43,
M+).
Protein Conjugates of Hapten 5 (23). The mixed anhydride was
formed using dry DMF with stirring under nitrogen. Hapten 5 (24.9
mg, 60 µmol) was dissolved in DMF (3.2 mL) and tributylamine (360
µL of 0.2 M solution in DMF, 72 µmol), then isobutyl chloroformate
(360 µL of 0.2 M solution in DMF, 72 µmol) was added dropwise to
the solution at 4 °C. The mixture was maintained at the same
temperature for 10 min and at room temperature for 20 min, then
divided into four equal portions. Each aliquot was added dropwise to
a solution of a carrier protein (15 mg of BSA, CONA, KLH, or OVA)
in 0.2 M borate buffer, pH 8.7 (5.0 mL) at 4 °C. The reaction mixtures
were stirred at the same temperature for 2 h, and at room temperature
overnight, and then the resulting conjugates were purified by exhaustive
dialysis in PBS. The conjugates of proteins BSA, CONA, KLH, and
OVA were designated as 23-BSA, 23-CONA, 23-KLH, and 23-OVA,
respectively; UV-Vis (PBS) 275-278. In the UV-Vis spectra of
hapten 5 and conjugates 23, the peaks were very close to those of the
carrier proteins; thus, it was not possible to estimate the hapten/protein
ratios of these conjugates from the UV-Vis spectral data.
4-Methyl-2-(2-phenoxyphenyl)azo-phenol (26a). A suspension of
2-phenoxyaniline (24a, 185.2 mg, 1 mmol) in 0.1 M HCl (25 mL) was
sonicated for 15 min. The diazotation and azo coupling reactions were
then carried out with stirring. A solution of aqueous sodium nitrite
(0.43 M, 2.4 mL, 1.03 mmol) was added to the amine at 4-5 °C in 15
min. The reaction mixture was kept at the same temperature for 30
min. Urea (12 mg, 0.2 mmol) was then added, and the mixture was
stirred for 5 min. Meanwhile, 4-methylphenol (25, p-cresol, 108.1 mg,
1 mmol) was dissolved in Borax solution (0.2 M, 15 mL), the solution
was cooled to 4 °C, and the diazonium salt solution was added to this
solution dropwise at 4-5 °C. The mixture was kept at 5 °C for 30 min
and at room temperature for 3 h. The precipitate was then collected by
filtration, washed with water and hexane, and recrystallized from
isopropyl ether to afford 248 mg (81%) of 26a as a crystalline product.
mp 67-68 °C. UV-Vis (methanol) 322 (ꢀ ) 21,200). IR (KBr) 1583,
1490, 1478, 1253, 1238, 810, 759. 1H NMR (CDCl3) 2.3 (s, 3 H), 6.88
(d, 1 H), 6.98-7.18 (m, 5 H), 7.3-7.5 (m, 4 H), 7.58 (d, 1 H), 7.68 (s,
1 H), 12.5 (s, OH).
RESULTS AND DISCUSSION
Synthetic Work. Spacer groups were linked to the fenoxy-
carb molecule (1, Figure 1) at several positions to obtain haptens
(2-5, Figures 2-5) that were then chemically activated to
prepare their protein conjugates. Selected protein derivatives
were used as synthetic immunogens (2) to elicit rabbit polyclonal
antisera recognizing fenoxycarb. Some protein conjugates were
used as microplate coating antigens in the ELISA procedure.
The structure of fenoxycarb (1) offers various possibilities for
spacer arm attachment (Figure 1). Amino derivatives at the
terminal and internal rings (haptens 2 and 3, respectively) were
used to generate the corresponding diazonium salts (Figures 2
and 3). The diazonium compounds were linked to proteins by
azo coupling (Figures 2 and 3). Elongation of the ethyl group
of the parent structure led to hapten 4 with a carboxylic acid
functional group (Figure 4). The nitrogen of the fenoxycarb
molecule was alkylated to obtain another carboxylic acid hapten
5 (Figure 5). Protein conjugates of the latter two compounds (4
and 5, linker group for both: (CH2)5CO2H) were obtained by
activation of the carboxyl groups.
Our hapten design was guided by the principle of maximizing
steric and electronic similarity with the parent molecule, as well
as by the feasibility of the syntheses (2, 38). Hapten synthesis
often comprises the most time-consuming and difficult part of
immunoreagent development (38). Thus, both the similarity
criteria and the burden of preparative effort have to be carefully
evaluated before undertaking the synthesis of a hapten. Synthesis
of a near-perfect analogue of the target analyte is sometimes
challenging. Such a high degree of structural similarity can,
4-Methyl-2-(3-phenoxyphenyl)azo-phenol (26b). This compound
was prepared in a similar fashion from 3-phenoxyaniline (24b). 239
mg (78%) of 26b as a crystalline product was obtained. mp 73-74

Development of ELISAs for IGR Fenoxycarb
J. Agric. Food Chem., Vol. 50, No. 1, 2002 35
Table 1. Characterization of Selected ELISA Systems
A
B
C
system
(homologous)
(homologous) (heterologous)
antiserum/immunogen
dilution in competitive
ELISA
39189a/20′′-KLH 4945/14-KLH 4945/14-KLH
1:1,000
1:3,000
1:1,500
coating antigen
concentration (µg/mL)
titer
20′′-BSA
5
1:50,000
14-BSA
1
1:65,000
102
16-BSA
2.5
1:20,000
17
IC50 (ppb of fenoxycarb) 95
fenoxycarb detection
13−2,000
12−3,000
0.5−300
range (ppb)
Figure 6. Syntheses leading to compounds 26a−c.
however, be necessary to make a good immunizing hapten (2).
In our synthetic work, preparation of hapten 2 (Figure 2) with
structure closely related to fenoxycarb required the most effort.
The ELISA (system C, Table 1) using the antiserum based on
this immunizing hapten (2), however, resulted in the highest
sensitivity in this study (see Competitive ELISAs below).
Synthetic precursor 13 of hapten 2 was assembled from the
tosilate 9, including the carbamate moiety of the fenoxycarb
molecule, and 4-(4-nitrophenoxy)phenol (12, Figure 2). The high
reactivity of fluorine in 1-fluoro-4-nitrobenzene (10) toward
nucleophiles (39, 40) enabled the development of a simple
preparation of the phenol 12, which was based on selective
substitution reaction of compound 10 with hydroquinone (11).
Such an approach can be a useful alternative to other methods
(36, 41) for the synthesis of diaryl ethers related to compound
12, if the reactive fluoroaromatic starting material is readily
available. Ethyl 2-hydroxyethylcarbamate (8) was obtained by
selective derivatization of ethanolamine (7) in the presence of
potassium fluoride. The role of fluoride ions was to enhance
reactivity of the amino group by disrupting strong hydrogen
bonds in the starting material (7) (42-44). This technique
allowed a simple nonaqueous workup. The conventional variant
of this reaction using aqueous base and extractive isolation gave
less satisfactory results. The tosilate 9, obtained from the alcohol
8, was condensed with the phenol 12 in the presence of
potassium carbonate. The nitro group of the resulting carbamate
13 was reduced by catalytic transfer hydrogenation (CTH)
employing the ammonium formate/palladium-on-carbon system
(37, 45). This method produced the corresponding amine (2) in
good yield under mild conditions without the difficulties
involved using hydrogen gas. The amine hapten 2 was then
diazotized in an aqueous solution following established proce-
dures (46-48). The azo coupling reaction (46-49) of the
resulting diazonium salt with proteins yielded the corresponding
conjugates (14).
Adaptation of a general literature procedure (35, 50) led to a
mild and selective nitration method for fenoxycarb (Figure 3).
Acetyl nitrate, formed from silver nitrate and acetyl chloride in
situ (50), served as nitrating agent. The more activated internal
ring of the fenoxycarb molecule was substituted selectively
(Figure 3). It appears that a mixture of the two isomeric nitro
compounds (15) was formed, but no efforts were made to isolate
the individual isomers (Figure 3). This mixture (15) was reduced
to the mixture of the corresponding amino compounds (3) by
the CTH procedure (see above) with, again, no attempt to isolate
the two individual isomers (Figure 3). The resulting hapten (3,
isomer mixture) was diazotized by both the routine aqueous
method (46-48) and a modified procedure using butyl nitrite
in DMSO (Figure 3). We devised the latter diazotation method
to conjugate a highly lipophilic dioxin derivative, weakly basic
and poorly soluble in water, to proteins (51). It was also
successfully applied to prepare conjugates of other lipophilic
compounds such as pyrethroid haptens (46). The diazonium salt,
formed from amine 3 by either method, was linked to proteins
by azo coupling to furnish the corresponding conjugates 16
(obtained via the routine diazotation method) and 16′ (furnished
by the modified procedure).
Following literature analogies (52), ethyl 6-hydroxyhexanoate
(17) was successively treated with 4-nitrophenyl chloroformate
(18) and 2-(4-phenoxyphenoxy)ethylamine (6) to produce the
carbamate intermediate 19 (Figure 4). Carbamate groups such
as that of compound 19 are usually fairly resistant to saponifica-
tion. Thus, selective alkaline hydrolysis of the ester group of
intermediate 19 to form hapten 4 could be performed in good
yield (Figure 4). This carboxylic acid (4) was conjugated to
proteins by the active ester method (1) to form conjugates (20,
20′, and 20′′) with several hapten/protein ratios (Figure 4).
The amide anion, generated from fenoxycarb (1) by sodium
hydride, was alkylated using ethyl 6-bromohexanoate (21) (53,
54) to yield the ester 22 (Figure 5). This intermediate (22) was
converted to hapten 5 by selective alkaline hydrolysis. The
resulting carboxylic acid (5) was coupled to proteins by the
mixed anhydride method (53) to furnish the corresponding
conjugates (23, Figure 5).
Diazotation of the three phenoxyaniline-isomers (24a-c,
Figure 6) and coupling of the resulting diazonium salts with
4-methylphenol (25) following standard protocols (47, 48) gave
26a-c model compounds for epitope density calculations (see
below). The lack of λOH signals in the IR-spectra and the very
1
high shifts of the OH signals in the H NMR-spectra of azo
derivatives 26a-c (Figure 6) are due to strong intramolecular
hydrogen bonds involving the hydroxyl and azo groups (48).
These unusual spectral properties of compounds 26a-c are
similar to those of related azo compounds with hydrogen-bonded
chelate structures (48).
Estimation of Hapten Densities (HDs) in Protein Conju-
gates. The colored protein derivatives 14, 16, and 16′, obtained
by azo coupling, display broad absorbance bands in the range
of about 270-600 nm. We have utilized the strong UV-Vis
absorbances due to azo moieties to calculate the approximate
hapten/protein ratios of these conjugates (14, 16, and 16′). Under
the conditions used in our azo coupling reactions (pH ∼9-
10), diazonium salts readily form azo bridges with tyrosines
and, to a lesser extent, a few other amino acids (e.g., histidine)
in proteins (49, 55, 56). Thus, our estimation of HDs is based
on molar extinction coefficient (ꢀ) values of model azo
compounds structurally related to the fenoxycarb-azo-tyrosine
moieties in the protein conjugates 14, 16, and 16′.
Because haptens 2 and 3 were available only in limited
amounts, we choose commercially available aromatic amines
(24a-c), structurally similar to these haptens, as starting
materials for syntheses of model compounds 26a-c (Figure 6).

36 J. Agric. Food Chem., Vol. 50, No. 1, 2002
Table 2. Results of Hapten Density Calculationsa,b
Szurdoki et al.
Table 3. Selectivity of the ELISA Systems toward Compounds Related
to Fenoxycarb
carrier
protein
number of
hapten
cross-reactivity (CR (%))a
λ
max of the molecular ꢀ used in the
b,c
b,c
b,c
compound
fenoxycarb (1)
2-(4-phenoxyphenoxy)ethylamine (6)
4-phenoxyphenol
system A
100
9.2
system B
system C
100
1.4
0.2
conjugate
(nm)
weight
(kDa)
calculation hapten molecules/10 kDa
(L/mol/cm) densityc carrier proteind
100
conjugate
d
−
−
20,150e
20,150e
20,150e
20,150e
20,150e
20,150e
20,150e
20,150e
20,150e
20,150e
28
19
28
8.4
25
20
714
43
8.9
75
4.2
2.5
0.84
1.9
3.8
2.6
1.2
1.3
2.1
1.1
3.5
1.1
2.2
2.4
d
d
16-BSA
16-CONA
16-LPH
16-OVA
16′-BSA
16′-CONA
16′-KLH
16′-LPH
16′-OVA
16′-TYG
14-BSA
14-KLH
14-OVA
14-TYG
396
394
392
393
399
396
397
398
395
397
358
378
355
360
67
76
335
43
67
76
6,000
335
43
660
67
6,000
43
660
−
a Results are expressed as percent cross-reactivity values. Cross-reactivity (CR)
value (%) ) (IC50 of fenoxycarb/IC50 of compound) × 100. b Assay systems were
as described in Table 1. c Methoprene and a variety of compounds (e.g.,
pyriproxyfen, 4-phenylphenol, 2-hydroxydibenzofuran, fenvalerate, cypermethryn,
2,4-D, acifluorfen, fluorodifen, p,p′-DDT) structurally related to fenoxycarb did not
produce significant inhibition (CR < 0.1%). d Significant inhibition was not observed
in these cases.
f
20,900
23
f
20,900
636
9.2
159
f
be somewhat higher due to azo coupling to a few amino acids
(e.g., histidine) beside tyrosine in the carrier proteins. In our
study, HDs for certain conjugate groups, each based on one of
the carrier proteins BSA, CONA, KLH, and OVA, are markedly
consistent between each group (Table 2). This trend is likely
because of some structural properties of the individual native
proteins. The hapten/10 kDa protein ratios (HD10kDa) allow
comparison between conjugates of different carrier proteins (53).
HD10kDa data range between 0.8 and 4.2, with the highest
values for conjugates of BSA and CONA (Table 2).
Antisera Characterization, Titer. The results of antibody
characterization were obtained from antisera of terminal bleeds.
Raw antisera were used without purification. Titer is defined
as the serum dilution giving three times the background
absorbance (60). Titers were determined by the usual checker-
board titrations (46). Most antisera showed fairly high titers to
their homologous antigens; typical reciprocal titers were on the
order of 103 to 105. Selected examples (systems A and B) are
shown in Table 1 and Figure 7. Some heterologous assays such
as system C (Table 1, Figure 7) also displayed promising titers.
Competitive ELISAs. First, homologous assay systems were
investigated in competition experiments. Selected assays (sys-
tems A and B, see Table 1 and Figure 8) detected fenoxycarb
in the range of 12 to 3,000 ng/mL (ppb). Heterology provides
the means of modifying equilibrium conditions in competitive
immunoassays (2, 53, 60). Thus, heterologous assays are often
used to enhance the sensitivity of ELISAs (2, 51, 53, 56, 60).
In our work, site and carrier heterology in system C resulted in
improved sensitivity (IC50, 17 ppb; LDL, 0.5 ppb; Table 1,
Figure 8) as compared to the homologous ELISA based on the
same antiserum (system B, Table 1). The high sensitivity of
system C using antiserum 4945, based on immunogen 14-KLH
and immunizing hapten 2 (Figure 2), may be also due to the
relatively short and rigid azo group spacer in conjugate 14-
KLH of the lipophilic analyte (1). Such a linker group attached
to the terminal ring of fenoxycarb may have helped to project
the fenoxycarb moieties on the immunogen (14-KLH, Figure
2) for recognition by preventing the haptens from folding back
on the adjacent lipophilic areas of the protein surface (51).
Cross-Reactivity. The assays showed no significant interfer-
ences (CR < 0.1%, Table 3) with 4-phenylphenol and 2-hy-
droxydibenzofuran, photodegradation products of fenoxycarb
(61, 62), which may be of advantage for environmental analysis
of fenoxycarb. Likewise, the terpenoid methoprene and aromatic
pyriproxyfen IGR insecticides did not cause significant inhibi-
tion with these ELISAs. The structures of the latter compound
and fenoxycarb share the 4-phenoxyphenoxy moiety. A variety
of further pesticides structurally related to fenoxycarb, including
20,900
f
20,900
a Protein concentrations of the conjugates were determined by the BCA protein
assay. b Conjugate 16-TYG was not involved in this study. c Average number of
hapten moleules linked to a carrier protein molecule; HD. d HD 10 kDa. e Average
of ꢀ values for 26a and 26b model compounds. f ꢀ value for 26c model compound.
In the structures of azo derivatives 26a-c, the fragment
according to 4-methylphenol (25) mimicks the phenolic group
of tyrosine (Figure 6). Our approach is similar to that of
Flanagan et al. (57), who determined epitope densities for protein
conjugates prepared by azo coupling. The ꢀ value for the azo
linkage, used by these authors, was that of an azo derivative
obtained by diazotation of an aromatic amine hapten and azo
coupling with 4-methylphenol (25) (57, 58). The ꢀ values at
the wavelengths of maximal absorbance (λmax) of model
compounds 26a-c are taken into account in our calculations
(see Materials and Methods and Table 2). The ring-substitution
pattern of azoaromatic groups in the structure of protein
conjugates 14 is analogous to that of compound 26c synthesized
from 1,4-phenoxyaniline (24c, Figures 2 and 6). Thus, the ꢀ
value of azo derivative 26c is used to estimate HDs of this set
of conjugates (14, Table 2). Conjugates 16 and 16′, based on
hapten 3, are structurally related to azo compounds 26a and
26b (Figures 3 and 6). Azo derivatives 26a and 26b were
synthesized from 1,2- and 1,3-phenoxyaniline (24a and 24b,
Figure 6), respectively. Because the two isomers, whose mixture
comprises hapten 3, are not identified, the average (20,150) of
ꢀ values for compounds 26a and 26b is employed in calculations
for 16 and 16′ sets of conjugates (Figures 3 and 6, Table 2).
Spectral characteristics (e.g., ꢀ and λmax values) of azo com-
pounds 26a and 26b are very close (see Materials and Methods).
The ꢀ values used in our calculations (Table 2) and reported in
the literature for azo derivatives structurally related to our model
compounds are similar (57, 59). The differences between the
λmax values of our model compounds 26a-c and those of the
corresponding protein conjugates (14, 16, and 16′) appear to
be due to structural differences between azo compounds 26a-c
and conjugates (14, 16, and 16′) and/or to solvent effect and
influence of the protein microenvironment. The molecular
weights (kDa) of carrier proteins used in the calculations are as
follows: BSA, 67 (49); CONA, 76 (53); KLH, 6,000 (53); LPH,
335 (46); OVA, 43 (49); TYG, 660 (46, 49).
Although this HD estimation method is based on ꢀ values of
model compounds with only simple structures (26a-c), the
obtained HDs (Table 2) are similar to those reported for other
protein conjugates obtained by azo coupling (56, 57). Our
calculations give only approximate HDs; the real values may

Development of ELISAs for IGR Fenoxycarb
J. Agric. Food Chem., Vol. 50, No. 1, 2002 37
Figure 7. Titration curves obtained by ELISA systems used in this study. Assay systems were as described in Table 1. Microplate wells treated with a
coating antigen were exposed to various dilutions of an antiserum. See Materials and Methods for further details. Each point represents the average of
the OD values at 492 nm, which were from three replicate wells on the same microplates. Coefficients of variation for all points fell in the range of
0.3−13.3%.
Figure 8. Fenoxycarb standard curves obtained by competitive ELISA systems used in this study. Assay systems were as described in Table 1.
Microplate wells treated with a coating antigen were exposed to solutions containing an antiserum and various concentrations of fenoxycarb. See
Materials and Methods for further details. Each point represents the average ± SD of the OD values at 492 nm, which were from three replicate wells
on the same microplate.
the pyrethroids fenvalerate and cypermethryn, the phenoxyacetic
acid herbicide 2,4-D, p,p′-DDT, and the nitrodiphenyl ether
herbicides acifluorfen and fluorodifen, also produced no sig-
nificant cross-reactivity (Table 3). 4-Phenoxyphenol (Table 3),
a likely metabolite of fenoxycarb in Heliothis Virescens (63),
showed no or only weak interference with the assays. The higher
cross-reactivity of ELISA system A than those of systems B
and C for 2-(4-phenoxyphenoxy)ethylamine (6, Figure 4, Table
3) can be interpreted in terms of the structures of the immunizing
haptens. The spacer arm in hapten molecule 4 is linked to the
ethyl group of fenoxycarb (Figure 4). The 4-phenoxyphenoxy
group is the immunodominant part of the corresponding
immunizing antigen (20′′-KLH, Figure 4) because of the
shielding effect of the carrier protein around the spacer group
attachment (2, 54). Thus, the resulting antiserum displays the
highest affinity toward the aromatic moiety of fenoxycarb
(system A, Table 3). The opposite tendency is expected for the
antiserum based on hapten 2 considering that the linker azo

38 J. Agric. Food Chem., Vol. 50, No. 1, 2002
Szurdoki et al.
group is attached to the terminal phenyl ring of fenoxycarb in
the immunogen structure (14-KLH, Figure 2, systems B and C
in Table 3). Taken together, the cross-reactivity results (Table
3) demonstrate that our ELISAs, based on immunogen haptens
possessing the entire target analyte structure (Figures 1, 2, and
4), are highly selective for fenoxycarb. It appears that an epitope
encompassing at least the 2-(4-phenoxyphenoxy)ethylamine (6)
molecule is required for potent inhibition of ELISAs A and C.
ELISA B displays a high degree of specificity for fenoxycarb
because the whole target analyte structure is necessary for strong
inhibition.
assistance with spectral studies. We thank I. Morera and M. A.
Miranda (Department of Chemistry, Polytechnic University of
Valencia, Valencia, Spain) and D. Cerf (Novartis Crop Protec-
tion) for providing samples for cross-reactivity investigations.
We also thank S. Dedos (Institute of Biology, N. C. S. R.
“Demokritos”, Athens, Greece), P. G. Schultz (Chemistry
Department, Scripps Research Institute, La Jolla, CA), and I.
Ujva´ry (Institute for Chemistry, Chemical Research Center,
Budapest, Hungary) for helpful discussions.
LITERATURE CITED
Conclusions. The sensitivity and selectivity of our heterolo-
gous assay are similar to those of the ELISA reported by Giraudi
et al. (28). The Italian group’s hapten-homologous assay is based
on the hapten 2-(4-phenoxyphenoxy)ethanol hemisuccinate,
includes only a part of the fenoxycarb structure (1), and purified
polyclonal antibodies (28). Their optimized ELISA is character-
ized with an IC50 of 9.5 ppb and an LDL of 0.42 ppb, and
displays significant interference with pyriproxyfen (CR, 6.0%).
The assay system of Giraudi et al. (28) and our highly
sensitive and selective ELISAs for fenoxycarb hold promise for
rapid and inexpensive analysis of large numbers of samples for
environmental and agricultural screening programs. These
immunoassays may also provide a useful analytical tool for
investigations on the biochemical mode of action and metabo-
lism of this compound (1). The results presented here, our
previous experience, and literature reports suggest that it is
usually rewarding to explore several haptens with different
spacer arm locations and sizes, various conjugation methods,
and carrier proteins during ELISA development (2, 46, 51, 53,
54, 56, 60). Further studies with other assay systems and real
samples are in progress.
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ABBREVIATIONS USED
Borax, Na2B4O7‚10 H2O; BSA, bovine serum albumin;
CONA, conalbumin; CR, cross-reactivity; CTH, catalytic trans-
fer hydrogenation; DCC, dicyclohexylcarbodiimide; DMF, N,N-
dimethylformamide; DMSO, dimethyl sulfoxide; EI, electron
impact; ELISA, enzyme-linked immunosorbent assay; EPA,
Environmental Protection Agency; GC, gas chromatography;
HD, hapten density (hapten/protein ratio); HD10kDa, hapten/
10 kDa protein ratio; IC50, analyte concentration required for
50% inhibition; IR, infrared spectroscopy; KLH, keyhole limpet
hemocyanin; LDL, lower detection limit; LPH, hemocyanin of
Limulus polyphemus; MS, mass spectrometry; MS-ES+, elec-
trospray MS in positive mode; NHS, N-hydroxysuccinimide;
NMR, nuclear magnetic resonance spectroscopy; OD, optical
density; OVA, ovalbumin; PBS, phosphate-buffered saline (0.01
M phosphate buffer + 0.8% NaCl, pH 7.5); PBST, PBS +
0.05% Tween 20 (pH 7.5); ppb, parts per billion (ng/mL); SD,
standard deviation; THF, tetrahydrofuran; TLC: thin-layer
chromatography; TYG, porcine thyroglobulin; UV-Vis, ultra-
violet-visible spectroscopy.
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Received for review May 31, 2001. Revised manuscript received October
10, 2001. Accepted October 11, 2001. Partial financial support was
provided by the NIEHS Superfund Basic Research Program P42
ES04699 and the NIEHS Center P30 ES05707. This work was also
supported by a collaborative grant of the European Union (EC INCO-
COPERNICUS ERBIC15CT960802), the US-Hungarian Joint Fund
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