Development of sensitive immunoassays for the detection of the glucuronide conjugate of 3-phenoxybenzyl alcohol, a putative human urinary biomarker for pyrethroid exposure

Article abstract of DOI:10.1021/jf063282g

Pyrethroids are widely used in agriculture as insecticides. This study describes a sensitive enzyme-linked immunosorbent assay for the detection of the glucuronide conjugate of 3-phenoxybenzyl alcohol, a putative pyrethroid metabolite that may be used as a biomarker of exposure to pyrethroids. Four antisera were elicited against two different immunizing haptens. Antisera were characterized in combination with several coating haptens. The lowest IC ₅₀ value (0.5 ng/mL) was obtained with antiserum 1891 and 3-phenoxybenzoic acid-BSA conjugate as the coating antigen. Antiserum 1891 was highly selective for the target compound with an overall cross-reactivity of <0.3% to structurally related compounds. The assay sensitivity was negligibly affected by pH 4-9. A 5-fold improvement in IC₅₀ was observed using a 10-fold concentrated phosphate-fuffered saline as the assay buffer. Compared to assays conducted in normal phosphate-fuffered saline, the maximal absorbance was almost identical. A good correlation (r² = 0.99 and 0.97 for urine samples A and B, respectively) was observed between spiked levels and the levels detected by the immunoassay.

Full text of DOI:10.1021/jf063282g

3750 J. Agric. Food Chem. 2007, 55, 3750−3757
Development of Sensitive Immunoassays for the Detection of
the Glucuronide Conjugate of 3-Phenoxybenzyl Alcohol, a
Putative Human Urinary Biomarker for Pyrethroid Exposure
HEE-JOO KIM,† KI CHANG AHN,† SEUNG JIN MA,§ SHIRLEY J. GEE,† AND
BRUCE D. HAMMOCK*,†
Department of Entomology and UCD Cancer Research Center, University of California,
Davis, California 95616, and Department of Food Engineering, Mokpo National University,
61 Dorim-ri, Cheonggye-myeon, Muaan-gun, Jeonnam, South Korea
Pyrethroids are widely used in agriculture as insecticides. This study describes a sensitive enzyme-
linked immunosorbent assay for the detection of the glucuronide conjugate of 3-phenoxybenzyl alcohol,
a putative pyrethroid metabolite that may be used as a biomarker of exposure to pyrethroids. Four
antisera were elicited against two different immunizing haptens. Antisera were characterized in
combination with several coating haptens. The lowest IC50 value (0.5 ng/mL) was obtained with
antiserum 1891 and 3-phenoxybenzoic acid-BSA conjugate as the coating antigen. Antiserum 1891
was highly selective for the target compound with an overall cross-reactivity of <0.3% to structurally
related compounds. The assay sensitivity was negligibly affected by pH 4-9. A 5-fold improvement
in IC50 was observed using a 10-fold concentrated phosphate-fuffered saline as the assay buffer.
Compared to assays conducted in normal phosphate-fuffered saline, the maximal absorbance was
almost identical. A good correlation (r 2 ) 0.99 and 0.97 for urine samples A and B, respectively)
was observed between spiked levels and the levels detected by the immunoassay.
KEYWORDS: ELISA; pyrethroid; glucuronide conjugate; human exposure; hapten
INTRODUCTION
Most commercial pyrethroids are esters. These pyrethroids
are metabolized rapidly by oxidation and hydrolytic cleavage
of the ester linkage to cis/trans-3-(2,2-dichlorovinyl)-2,2-
dimethylcyclopropane-1-carboxylic acid (DCCA) and 3-phe-
noxybenzyl alcohol (Figure 1). The 3-phenoxybenzyl alcohol
is further processed to 3-phenoxybenzoic acid (PBA). DCCA
and both the benzyl alcohol and the benzoic acid may undergo
further metabolism to glucuronide, glycine, taurine, and sulfate
conjugates (13-18). The cyanobenzyl alcohol from compounds
such as cypermethrin and fenvalerate is expected to rearrange
chemically to the corresponding benzoic acid (PBA), and thus
the biomarker developed here will not be useful for type II
pyrethroids. However, the glucuronide conjugate of 3-phenoxy-
benzyl alcohol (3-PBAlc-Gluc) should be a useful biomarker
for exposure to type I pyrethroids such as phenothrin and
permethrin that contain a 3-phenoxybenzyl alcohol moiety.
Pyrethroids exert neurotoxic effects on the axons of the
nervous system by interacting with sodium channels in insects
and mammals (1). High potency in controlling a wide spectrum
of insects and low toxicity to birds and mammals have made it
widely accepted for application in agriculture, forestry, homes,
horticulture, and public health around the world (2-4). Although
pyrethroids are considered to be safe for humans, there have
been concerns arising from their potential effects on human
health from long-term exposure or high exposure of children.
Environmental concerns include accumulation and leaching into
the surface water and groundwater (5-8). Some research
indicates that high exposure to pyrethroids might cause endo-
crine disruption, suppressive effects on the immune system,
lymph node and splenic damage, and carcinogenesis (9-11).
Pyrethroids are also known to be highly toxic to some aquatic
species and beneficial insects. Pyrethroids have been found in
various agricultural and commercial products such as vegetables,
fruits, and shampoo and surface water and groundwater (12).
Therefore, it is important to develop a rapid, sensitive, and
efficient analytical method for both toxicological and epide-
miological monitoring.
Currently HPLC and GC-MS are the primary methods used
to analyze urinary metabolites of pyrethroids (19-22). Although
these methods provide accurate and reliable results, somewhat
complicated sample preparation cannot be avoided. Immunoas-
say has proven to be a sensitive analytical method for clinical
diagnostics, agriculture, environmental monitoring, and food
quality assessment. Previously we reported several immunoas-
says for the detection of pyrethroid parent compounds (23-
27), their primary metabolites, and various conjugates (28-
30). Although no study has specifically determined the exact
† University of CaliforniasDavis.
§ Mokpo National University.
10.1021/jf063282g CCC: $37.00
© 2007 American Chemical Society
Published on Web 04/25/2007

Immunochemistry Symposium
J. Agric. Food Chem., Vol. 55, No. 10, 2007 3751
Figure 1. Mammalian metabolism of the type I pyrethroid permethrin. Conjugates vary by species, but the relative abundance of various conjugates for
humans has not been determined.
Quattro Ultima triple-quadrupole tandem mass spectrometer (Micro-
mass, Manchester, U.K.). Melting points were determined on a Thomas-
Hoover Uni-Melt apparatus (Thomas Scientific, Swedesboro, NJ) and
are uncorrected. ELISAs were performed on 96 well microtiter plates
(Nunc-Immunoplate, MaxiSorp surface, Roskilde, Denmark) and read
spectrophotometrically with a microplate reader (Molecular Devices,
Menlo Park, CA) in dual wavelength mode (450-650 nm).
conjugates of pyrethroid metabolites in humans, the development
of immunoassays for all types of metabolites enhances the
capability of biological monitoring for an exposure to pyre-
throids because the pyrethroid metabolite profile will vary from
individual to individual, and analysis for a single metabolite
will not give a complete picture. In this study, we report a
polyclonal antibody-based enzyme-linked immunosorbent assay
(ELISA) for the detection of the putative urinary biomarker of
pyrethroid exposure, 3-PBAlc-Gluc. We describe the synthesis
of the glucuronide target analyte, hapten preparation using
heterobifunctional cross-linkers, antibody production against the
target conjugate, assay development, and validation with urine
samples.
Synthesis of 3-Phenoxylbenzyl â-D-Glucuronide (3-PBAlc-Gluc)
(4; Figure 2). Methyl 1,2,3,4-tetra-O-acetyl-D-glucopyranouronate (1;
7 g, 18.6 mmol) prepared from glucuronolactone by methylation with
sodium methoxide and acid-catalyzed acetylation with acetic anhydride
was dissolved in dry CH2Cl2 (100 mL), and 45% hydrobromic acid in
acetic acid (20 mL, 111.6 mmol) was added at ice temperatures. After
stirring at room temperature for 3 h, the mixture was diluted with CHCl3
and successively washed with saturated aqueous NaHCO3 and saturated
aqueous NaCl. The organic layer was filtered through activated charcoal
to remove the color. After evaporation, the residue was recrystallized
with ethyl ether to afford methyl (2,3,4-tri-O-acetyl-R-D-glucopyranosyl
bromide)uronate (2; 5.6 g, 76%). For the Koenigs-Knorr reaction, to
a solution of 2 (500 mg, 1.3 mmol) in dry CH2Cl2 (10 mL) were added
3-phenoxybenzyl alcohol (350 mg, 1.8 mmol), dry silver trifluo-
romethanesulfonate (400 mg, 1.6 mmol), and tetramethylurea (180 mg,
1.6 mmol), and the mixture was stirred under an argon atmosphere at
-20 °C for 12 h. The reaction was filtered and diluted with ethyl acetate
(EtOAc). The organic layer was washed with saturated aqueous
NaHCO3 and applied to flash chromatography (stepwise elution with
hexane/EtOAc; 20, 30, and 40% EtOAc) to afford 3-phenoxybenzyl
methyl 2,3,4-tri-O-acetyl-D-glucopyranouronate (3; 350 mg, 55%).
Compound 3 (250 mg, 0.48 mmol) was dissolved in methanol (MeOH,
5 mL), and a 1 M sodium methoxide solution (10 mL) was added. The
mixture was stirred at room temperature for 24 h, and an excess of
Ba(OH)2 was added. The reaction mixture was stirred at room
temperature for 24 h. After neutralization with Dowex-50 (H+-form),
the mixture was filtered and evaporated in vacuo. The residue was
purified with silica gel column chromatography (stepwise elution with
CHCl3/MeOH; 10, 20, and 30% MeOH) and with ODS column
MATERIALS AND METHODS
Chemicals. Organic chemicals for the syntheses were purchased from
Aldrich Chemical Co. (Milwaukee, WI) and Fisher Scientific (Pitts-
burgh, PA). Thin-layer chromatography (TLC) utilized 0.2 mm
precoated silica gel 60 F254 on glass plates from E. Merck (Darmstadt,
Germany). Column chromatographic separations were carried out using
Baker silica gel (40 µm average particle size) using the indicated
solvents. Heterobifunctional cross-linking reagents, (3-[2-aminoethyl]-
dithio)propionic acid‚HCl (AEDP) and (N-[ꢀ-trifluoroacetylcaproyloxy]-
succinic ester (TFCS), were purchased from Pierce (Rockford, IL).
Other coupling reagents were purchased from Aldrich. Bovine serum
albumin (BSA), thyroglobulin (Thyr), goat anti-rabbit IgG conjugated
to horseradish peroxidase (GAR-HRP), Tween 20, and 3,3′,5,5′-
tetramethylbenzidine (TMB) were purchased from Sigma (St. Louis,
MO).
Instruments. 1H nuclear magnetic resonance (NMR) spectra of
compounds synthesized were obtained on a General Electric QE-300
spectrometer (Bruker NMR, Billerica, MA) using tetramethylsilane as
an internal standard. Electrospray mass spectra in the positive (MS-
ESI+) or negative (MS-ESI-) mode were recorded on a Micromass

3752 J. Agric. Food Chem., Vol. 55, No. 10, 2007
Kim et al.
Figure 2. Scheme for synthesis of 3-PBAlc−glucuronide.
Figure 3. Conjugation route of 3-PBAlc−glucuronide to the protein (thyroglobulin or BSA) through the heterologous bifunctional linkers
chromatography (stepwise elution with acetonitrile/H2O; 20, 50, and
80% acetonitrile). Freeze-drying gave analytically pure 3-phenoxybenzyl
â-D-glucuronide (4) as a white powder (70 mg, 40%; total yield from
1, 17%): 1H NMRH (CD3OD), δ 3.26-3.63 (2H, Gluc H-4 and 5),
3.64 (1H, dd, J ) 7.8 and 6.3 Hz, Gluc H-3), 4.41 (1H, dd, J ) 7.8
and 6.3 Hz, Gluc H-2), 4.67 (1H, dd, J ) 11.7 and 5.1 Hz, OCHHa-
Ph), 4.87 (1H, d, J ) 7.8, Gluc H-1), 5.01 (1H, dd, J ) 11.7 and 5.1
Hz, OCHbHPh), 6.92-7.40 (9H, -Ph-O-Ph); HR-neg-MS, m/z [M
- H]+ calcd for C19H19O8, 375.1080; found, 375.1052.
Preparation of Immunogens and Coating Antigens. 3-PBAlc-
Gluc was coupled to proteins to yield hapten-protein conjugates for
immunizing and coating antigens using two heterobifunctional coupling
reagents. Each heterobifunctional cross-linker possesses two different
reactive groups that allow for sequential conjugations with specific
functional groups of proteins. The resulting conjugates were similar
except that the acid moiety of 3-PBAlc-Gluc was linked through either
a -NHCH2SS(CH2)2CO- or -NH(CH2)5CO- spacer by using AEDP
or TFCS, respectively (Figure 3). 3-PBAlc-Gluc-AEDP or 3-PBAlc-
Gluc-TFCS was conjugated to Thyr for use as immunogen. For plate
coating antigens, both haptens (cAg01, cAg02) and PBA (cAg05) were
coupled to BSA.
Cross-Linking of 3-PBAlc-Gluc to Protein by AEDP. N-Hydroxy-
succinimide (NHS) (0.06 mmol) and dicyclohexylcarbodiimide (DCC)
(0.05 mmol) were added to 3-PBAlc-Gluc (0.04 mmol) dissolved in
0.2 mL of dry dimethylformamide (DMF). After the mixture had been
stirred overnight at 4 °C under a N2 atmosphere, the precipitated
dicyclohexylurea was removed by filtration. To the activated ester
solution (about 0.2 mL) were added AEDP (0.04 mmol) and triethyl-
amine (0.04 mmol). The mixture was allowed to react at 4 °C overnight.
After 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC, 0.08 mmol) in 0.5 mL DMF was added to the mixture and
reacted at 4 °C overnight, the reaction mixture was added slowly to
the protein solution (25 mg of protein in 5 mL of 0.05 M borate buffer
at pH 8) with vigorous stirring and then allowed to stir gently at 4 °C
for 24 h to complete the conjugation. This was followed by exhaustive
dialysis against normal strength phosphate-buffered saline.

Immunochemistry Symposium
J. Agric. Food Chem., Vol. 55, No. 10, 2007 3753
Cross-Linking of 3-PBAlc-Gluc to Protein by TFCS. TFCS (0.04
mmol) dissolved in 0.5 mL of DMF was added to the protein solution
(25 mg in 5 mL of 100 mM sodium phosphate, 0.15 M NaCl, pH 7.2).
After the mixture had been stirred at 4 °C for 2 h, the pH was adjusted
to pH 7.8-8.1 with diluted 1 N NaOH and allowed to incubate for 2
h to remove the trifluoroacetyl protecting group of the cross-linker.
The NHS-activated ester of 3-PBAlc-Gluc (0.04 mmol) was slowly
added to the linker-attached protein solution. The reaction mixture was
purified as described above.
Conjugation of PBA to Protein. NHS (0.06 mmol) and DCC (0.05
mmol) were added to PBA (0.04 mmol) dissolved in 0.2 mL of dry
DMF. The activated ester of PBA was added to a BSA solution (25
mg in 5 mL of 0.05 M borate buffer at pH 8) as described above.
Hapten Density Analyses. For the determination of the amount of
hapten (3-PBAlc-Gluc-linker) conjugated to protein, the BSA
conjugate was dialyzed against distilled water for 24 h to remove salts,
and then a powder was obtained by lyophilization. Hapten densities of
the BSA conjugates were determined by matrix-assisted laser desorption
ionization time-of-flight mass spectrometry (MALDI-TOF-MS) by
comparing the molecular weight of the standard BSA to that of the
conjugates. MALDI spectra were obtained by mixing 1 µL of matrix
(E-3,5-dimethoxy-4-hydroxycinnamic acid, 10 mg/mL) and 1 µL of a
solution of the conjugates (5 mg/mL in 5% formic acid)
Immunization and Antiserum Preparation. Two female New
Zealand white rabbits were immunized for each immunogen. Each
immunogen (3-PBAlc-Gluc-AEDP-Thyr or 3-PBAlc-Gluc-TFCS-
Thyr, 100 µg) in 0.5 mL of 0.85% saline was emulsified with an equal
volume of Freund’s complete adjuvant, and then the emulsion was
injected subcutaneously. After 3 weeks, the animals were boosted with
an additional 100 µg of immunogen that was emulsified with Freund’s
incomplete adjuvant (1:1, v/v). The boosts were given every 3 weeks,
and blood samples were drawn 7 days after each boost to check the
titers of antisera. The final antisera were collected 5 months following
the first immunization. The blood was collected into a Vacutainer tube
with a serum separation gel. The antisera were obtained by centrifuga-
tion and stored at -80 °C. The antiserum was used without further
purification.
ELISA Buffer Solutions. Normal strength PBS (1× PBS; 8 g/L of
NaCl, 0.2 g/L Na2HPO4, and 0.2 g/L of KCl, pH 7.5), PBST (PBS
containing 0.05% Tween 20), carbonate-bicarbonate buffer (1.59 g/L
Na2CO3, 2.93 g/L NaHCO3, pH 9.6), and 0.05M citrate-acetate buffer
(14.71 g/L Na3C6H5O7‚2H2O, pH 5.5) were used for immunoassay.
ELISA. Indirect competitive ELISAs were performed. The 96-well
microtiter plates were coated overnight at 4 °C with 100 µL/well of
the appropriate concentration of coating antigen (cAg) in 0.1 M
carbonate-bicarbonate buffer (pH 9.6). After it had been washed five
times with PBST, the plate was incubated with 200 µL/well of a 1.0%
BSA solution in PBS for 1 h at room temperature. After another washing
step, 100 µL/well of antiserum diluted in PBST per well (for titration
experiment) or 50 µL/well of antiserum diluted in PBST and 50 µL/
well of standard analyte or sample solution were added and incubated
for 1 h at room temperature. After the plate had been washed, 100
µL/well of the secondary GAR-HRP (1:6000 in PBST) was added
and incubated for 1 h at room temperature. The plate was washed again,
and 100 µL/well of a substrate solution [0.1 mL of 1% hydrogen
peroxide and 0.4 mL of 0.6% of TMB in dimethyl sulfoxide (DMSO)
added to 25 mL of citrate-acetate buffer, pH 5.5] was added to each
well. After 15 min at room temperature, the reaction was stopped by
adding 50 µL/well of 4 N H2SO4. The absorbance was measured using
a dual-wavelength mode at 450 minus 650 nm. Standard curves were
obtained by plotting absorbance against the logarithm of analyte
concentration, which was fit to a four-parameter logistic equation.
Assay Optimization. The assay conditions were optimized in such
a way that the IC50 values were minimized. This goal was achieved by
screening antibodies and antigens in a two-dimensional titration for
optimal dilution of cAg and antiserum. Then, competitive inhibition
curves were obtained for different antiserum and cAg combinations,
and the combination with the lowest IC50 was selected for further assay
development.
structurally related compounds (listed in Table 3). The CR was obtained
by comparing the IC50 values of the 3-PBAlc-Gluc standard to the
tested compounds, where %CR ) (IC50 of 3-PBAlc-Gluc/IC50 of tested
compound) × 100.
RESULTS AND DISCUSSION
Synthesis of Target Analyte and Hapten. It is known that
the main detoxification reaction of carboxylic acid containing
xenobiotics is conjugation either with an amino acid to form a
peptide or with glucuronic acid to form a glucoside. In this
respect, although the predominant type of conjugation for
metabolites of pyrethroids has not been elucidated, 3-phenoxy-
benzoic acid is known as a main metabolite of pyrethroids and
has been found in the general population at levels around 3
ppb (31). This metabolite arises from R-cyanopyrethroids by
rearrangement of the corresponding cyanohydrin. It also can
arise from PBAlc-containing pyrethroids by a two-step oxidation
of PBAlc to PBA (Figure 1). PBA can be conjugated as a
glucuronide, but acid glucuronides are less stable than alcohol
glucuronides and also tend to transesterify around the glucu-
ronide ring. We targeted the glucuronide of PBAlc as a possible
biomarker which could distinguish among pyrethroids that
hydrolyze primarily to PBAlc, PBA (ones with R-cyano
moieties), or other alcohol moieties. Alcohol glucuronides are
generally stable conjugates in the absence of glucuronidases.
Thus, the development of a specific assay for each possible
conjugate may be ideal for exposure monitoring.
The primary goals of this study were to synthesize the
glucuronide target compound and develop an immunoassay
specific to the 3-PBAlc-Gluc conjugate. The target compound
was first enzymatically synthesized with uridine 5′-diphospho-
glucuronic acid trisodium salt (UDPGA) and 3-PBAlc as the
substrate for glycosyl-S-transferase in mouse microsomes.
Because the yield was low from the enzymatic synthesis, a
chemical synthetic approach was taken. The chemical synthesis
was based on the methods of Bulgianesi and Shen (32) and
Sone and Misaki (33). The target analyte, 3-phenoxybenzyl â-D-
glucuronide, was synthesized as shown in Figure 2. The
Koenigs-Knorr reaction was used in the glucuronidation
between the O-protected methyl D-bromoglucopyronurate and
3-phenoxybenzyl alcohol via its silver salt. For the Koenigs-
Knorr reaction, 1-bromo derivatives of the glucuronic ester are
necessary as common intermediates.
To generate a specific antiserum for an analyte, the site for
the linker attachment must be selected to maintain the unique
target structure as distal as possible from the linker site while
minimizing structural conformation changes from the modifica-
tion. The phenyl ring is a potential site for linker attachment.
However, attachment at this site may result in antibodies with
high CR to other urinary phenolic glucuronides. Because the
phenoxybenzyl ring is the unique structural feature of the main
metabolite and because the high polarity of the glucuronide may
elicit a strong interaction for antibody recognition, the carboxylic
acid on the glucuronide was finally selected for the linker
introduction. Direct conjugation of the carrier proteins to the
carboxylic acid may not allow the glucuronide to be easily
accessible for antibody recognition, possibly due to steric
hindrance caused by bulky carrier proteins. This hypothesis is
supported by our observation that the antisera against p-ami-
nophenyl-â-D-glucuronide-Thyr without a linker space between
the hapten and carrier protein had very high IC50 values (240-
400 ng/mL) (34). Therefore, we used two types of commercial
cross-linkers, AEDP and TFCS, to provide space between the
hapten and carrier proteins (Figure 3). These cross-linkers
Cross-Reactivity (CR). The optimized assay was assessed for cross-
reactivity by using standard solutions of the analytes and other

3754 J. Agric. Food Chem., Vol. 55, No. 10, 2007
Table 1. Titration Summary for Anti-3-PBAlc Gluc Antisera
Kim et al.
−
a The data shown are at a cAg concentration of 1
0.7; +++, absorbance 0.7 1.0; ++++, absorbance > 1.0. Antisera 1890 and 1891 are derived from 3-PBAlc
3-PBAlc Gluc TFCS Thyr. Coating antigens are derived from conjugation of the hapten shown to BSA.
µ
g/mL and an antiserum dilution of 1:20000;
−
, absorbance < 0.2;
+
, absorbance
) 0.2−0.5; ++, absorbance )
0.5−
)
−
−Gluc−
AEDP Thyr and antisera 1892 and 1983 from
−
−
−
−
contain a primary amine functional group that reacts with the
carboxylic acid in the target molecule and a carboxylic acid
group that reacts with the lysine residues in the protein. We
used an active ester method to generate the amide linkage
between the hapten and the protein because of the precise control
and high yield of the reaction and to avoid raising antibodies
to protein-urea complexes, which sometimes result from the
use of water-soluble carbodiimides.
The MALDI-TOF-MS results showed the successful conjuga-
tion of 3-PBAlc-Gluc to BSA with the two linkers resulting
in molar ratios of hapten to BSA of 23:1 and 15:1 for 3-PBAlc-
Gluc-AEDP and 3-PBAlc-Gluc-TFCS, respectively.
Titers and Screening of the Antisera. Four antisera were
obtained against two immunizing haptens (sera 1890 and 1891
from 3-PBAlc-Gluc-AEDP and sera 1892 and 1893 from
3-PBAlc-Gluc-TFCS). A checkerboard titration method was
used to screen different combinations of those antisera with the
various coating antigens (cAgs). As shown in Table 1,
homologous combinations (cAg01 and cAg02 with antisera)
showed higher titers than heterologous combinations (cAg03,
cAg04, and cAg05 with antisera) due to the structural homology
of cAgs with the immunizing antigens. All antisera showed high
titers for cAg02, whereas antisera 1892 and 1893 showed very
low binding recognition for cAg01. For other tested cAgs with
the phenoxy moiety, antisera 1890 and 1891 showed measurable
recognition. However, the other two sera had negligible binding.
This result indicates that antisera appear to mainly bind the
phenoxy moiety of the compounds. Thus, antisera 1890 and
1891 were selected for further screening. Competitive binding
of antisera 1890 and 1891 against the free target analyte was
evaluated with each individual cAg. Although there was no
statistically significant difference in IC50 values between sera

Immunochemistry Symposium
J. Agric. Food Chem., Vol. 55, No. 10, 2007 3755
Table 2. Selected Competitive ELISA Results
IC50 (C)a
(ng/mL)
antiserum/cAg
ABSmax (A)
slope (B)
ABSmin (D)
1891/cAg01
1891/cAg02
1891/cAg03
1891/cAg04
1891/cAg05
1890/cAg01
1890/cAg02
1890/cAg03
1890/cAg04
1890cAg05
0.5
0.6
0.8
0.8
0.5
0.4
0.4
0.6
0.6
0.8
0.7
0.6
0.8
0.6
0.8
0.7
0.7
0.8
0.6
0.6
5.0
9.5
2.0
1.7
1.3
25.6
24.6
2.0
4.4
6.2
0.08
0.07
0.3
0.2
0.04
0.1
0.08
0.4
0.3
0.01
a Each value represents the mean value of four replicates.
Table 3. Summary of CR of 3-PBAlc−Gluc Assay
chemical
IC50 (ng/mL)
CR (%)
3-PBAlc
cypermethrin
permethrin
esfenvalerate
cyfluthrin
trans-DCCA
−
glucuronide
1.5
>2500
>2500
>2500
>2500
>2500
>2500
>2500
>2500
934
100
<0.06
<0.06
<0.06
<0.06
<0.06
<0.06
<0.06
<0.06
0.16
cis-DCCA
trans-DCCA glycine
p-nitrophenyl glucuronide
PBA
4-hydroxybenzoic acid
PBA−glycine
>2500
705
<0.06
0.21
1890 and 1891 for all cAgs, in general, the trend showed that
heterologous assays had relatively lower IC50 values than
homologous systems. More sensitive assays were obtained with
antiserum 1891 than with antiserum 1890. Among the five tested
cAgs, the most sensitive assay was obtained using cAg05 and
antiserum 1891 (IC50 of 1.3 ng/mL). For both antisera, cAg03
and cAg04 showed good sensitivity but also a high background
signal. Thus, cAg05 was finally selected for coating (Table 2).
CR. CR was evaluated with four parent pyrethroids (cyper-
methrin, permethrin, esfenvalerate, and cyfluthrin), their me-
tabolites (trans-DCCA, cis-DCCA, trans-DCCA-glycine, PBA,
and PBA-glycine), and structurally related compounds (p-
nitrophenyl glucuronide and 4-hydroxybenzoic acid). Antiserum
1891 is highly selective for the target compound with negligible
recognition of the other tested compounds (CR < 0.3%).
Although the phenoxybenzyl group appears to be necessary for
antibody recognition, an extremely low CR for PBA and
p-nitrophenyl glucuronide indicates that the phenoxybenzyl and
glucuronide are both required for a high binding of antiserum
1891 (Table 3).
Matrix Effects. High tolerance of an assay to the changes
in pH and ionic strength is desirable because this assay is
developed for direct detection of the metabolite in urine. Assay
performance under various pH and ionic strength conditions was
determined (Figure 4A). pH had little affect on the assay
sensitivity. Compared to pH 7.0, an approximately 10% sup-
pression of antibody binding was observed at pH 4, 5, and 6.
No changes in maximal absorbance were observed at pH 8 and
9. Although a 2-fold lower IC50 value was observed at pH 7.0,
this was not statistically significantly different. This indicates
that changes of pH in the tested range would not affect the
accuracy for the quantitation of the target compound. The assay
was highly tolerant to changes in ionic strength (Figure 4B).
In fact, as the ionic strength increased the IC50 decreased
compared to the IC50 (2.5 ng/mL) at 1× PBS. Sensitivity was
Figure 4. ELISA competition curves of 3-PBAlc
(A) pH values, (B) ionic strengths, and (C) percentages of urine. Reagent
concentrations were as follows: cAg, 0.25 g/well of PBA-BSA; 1:4000
final dilution, Ab 1891; and GAR HRP, 1:6000.
−Gluc prepared at various
µ
−
5-fold lower at 10× PBS. The antibody maintained 85% binding
capability so that thereafter assays were performed with 10×
PBS containing 0.05% Tween 20. This effect of ionic strength
could be due to a better buffering capacity of 10× PBS.
The effect of urine as a matrix was evaluated (Figure 4C).
In tests with four different urine samples, the assay parameters
were unchanged at 5% urine, but the antibody binding was
slightly suppressed at dilutions of 10 and 20%. Nevertheless,
the IC50 values were almost identical. Thus, to use urine samples

3756 J. Agric. Food Chem., Vol. 55, No. 10, 2007
Kim et al.
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Received for review November 13, 2006. Revised manuscript received
January 30, 2007. Accepted March 9, 2007. This research was supported
in part by the NIEHS Superfund Basic Research Program P42 ES04699,
NIEHS R37 ES02710, the NIEHS Center for Environmental Health
Sciences P30 ES05707, the NIOSH Center for Agricultural Disease and
Research, Education and Prevention 1 U50 0H07550, the Department
of Defense U.S. Army Medical Research and Materiel Command
Contract DAMD17-01-1-0679, and the NIEHS Center for Children’s
Environmental Health and Disease Prevention, P01 ES11269.
JF063282G