Highly sensitive enzyme-linked lmmunosorbent assay for chlorpyrifos. Application to olive oil analysis

Article abstract of DOI:10.1021/jf0516641

Highly sensitive enzyme-linked immunoassays for chlorpyrifos, one of the most applied insecticides, are presented. Several haptens were synthesized for immunoreagent production and ELISA development. The best immunoassays obtained are based on an indirect coated-plate immunoassay format. Two assays were optimized; one shows a limit of detection of 0.3 ng L^-1, an I ₅₀ of 271 ng L^-1, and a dynamic range between 4 and 16 474 ng L^-1. The other one has a limit of detection of 0.07 ng L ^-1, an I₅₀ of 7 ng L^-1, and a dynamic range between 0.4 and 302 ng L^-1. The assays were used to quantify chlorpyrifos in olive oil using a simple and rapid sample extraction procedure. The good recoveries achieved in both assays (109% mean value) and the agreement with values given by the GC reference method (110% mean value) indicate their potential for either screening or laboratory quantification.

Full text of DOI:10.1021/jf0516641

9352 J. Agric. Food Chem. 2005, 53, 9352−9360
Highly Sensitive Enzyme-Linked Immunosorbent Assay for
Chlorpyrifos. Application to Olive Oil Analysis
EVA M. BRUN, MARTA GARCEÄ S-GARCIÄA, ROSA PUCHADES,* AND
AÄ NGEL MAQUIEIRA*
Departamento de Qu´ımica, Universidad Polite´cnica de Valencia, Camino de Vera s/n,
46071 Valencia, Spain
Highly sensitive enzyme-linked immunoassays for chlorpyrifos, one of the most applied insecticides,
are presented. Several haptens were synthesized for immunoreagent production and ELISA
development. The best immunoassays obtained are based on an indirect coated-plate immunoassay
format. Two assays were optimized; one shows a limit of detection of 0.3 ng L-1, an I50 of 271 ng
L-1, and a dynamic range between 4 and 16 474 ng L-1. The other one has a limit of detection of
0.07 ng L-1, an I50 of 7 ng L-1, and a dynamic range between 0.4 and 302 ng L-1. The assays were
used to quantify chlorpyrifos in olive oil using a simple and rapid sample extraction procedure. The
good recoveries achieved in both assays (109% mean value) and the agreement with values given
by the GC reference method (110% mean value) indicate their potential for either screening or
laboratory quantification.
KEYWORDS: Organophosphorus pesticides; chlorpyrifos; ELISA; olive oil
INTRODUCTION
Chlorpyrifos is one of the insecticides used to treat one of
the most serious pests of olives in the Mediterranean basin
(Prays oleae, the olive moth), and the Codex Alimentarius
Commission of the Food and Agriculture Organization of the
United Nations (FAO) and the World Health Organization
(WHO) have established maximum pesticide residue limits in
olives and olive oil (10). Olive Oil Pesticide Residue Regulatory
Programs are being carried out to update and set new and more
restrictive regulations concerning the maximum residue levels
in these commodities. Analytical problems associated with the
determination of pesticide residues in olive oil are due to its
hydrophobic nature (11), so they are analyzed by chromato-
graphic techniques after tedious extraction and cleanup steps
(12). Sample preparation is a crucial stage in the analytical
procedure, since even small amount of lipids can harm the gas
chromatographic injectors and capillary columns and detectors,
which lead to losses in efficiency and sensitivity.
One of the most used organophosphorus pesticides is chlo-
rpyrifos, a nonsystemic chlorinated organophosphate insecticide,
developed by Dow Chemical Co. in 1962. It is applied world-
wide for agricultural crops protection and livestock healthcare
(1). The U.S. Environmental Protection Agency (USEPA)
estimates that 10 million pounds of chlorpyrifos are applied in
United States agriculture every year, half of which is used on
corn crops (2). It is also widely used in extensive and intensive
European agriculture, including crop fruits such as citrus, where
it is applied at a rate of over 50 000 kg per year (3). Until very
recently it was utilized worldwide in homes for pest control,
but some uses of this organophosphorus have been restricted
or eliminated (4).
Residues of chlorpyrifos in soils occur by direct application
or through spray drift/foliar washoff. This organophosphorus
is highly toxic to fish (acute LC50 values range from less than
1 mg L-1 to more than 200 mg L-1) and wildlife (5, 6). There
are no data suggesting that chlorpyrifos is a human carcinogen
(7), although it is a suspected endocrine disruptor (8). According
to these data, chlorpyrifos has been identified as a candidate
for priority review under the National Registration Authority’s
Existing Chemical Review Program by the U.S. National Drugs
and Poisons Schedule Committee in 2000 (9). Moreover, it is
in the Global Monitoring for Environment and Security/Food
Europe Comprehensive list of priority contaminants and com-
modity combinations.
Different chromatographic methods for the determination of
chlorpyrifos residues and its main metabolites in food and related
matrixes have been developed (13-18). Although chromato-
graphic techniques provide low limits of detection (0.001-0.1
µg L-1 in water), preliminary treatment (extraction, cleanup,
preconcentration, etc.) of the sample typically remains a man-
datory requirement, which makes the procedure laborious.
Immunoassay-based techniques, such as enzyme-linked im-
munosorbent assay (ELISA), can be simpler than conventional
chromatographic analysis, especially if a rapid extraction pro-
cedure is used. Immunoassay is also a good alternative to
chromatographic methods for monitoring a single defined target
when the multiresidue application is not justified.
* Corresponding author. Phone: +34-96-387 73 42. Fax: +34-96-387
93 49. E-mail: amaquieira@qim.upv.es (A.M.), rpuchades@qim.upv.es
(R.P.).
10.1021/jf0516641 CCC: $30.25
© 2005 American Chemical Society
Published on Web 10/28/2005

Sensitive Enzyme-Linked Immunosorbent Assay for Chlorpyrifos
J. Agric. Food Chem., Vol. 53, No. 24, 2005 9353
Several sensitive and specific ELISAs for chlorpyrifos have
been reported to detect it in primary matrixes (19-24). Using
different haptens, polyclonal (19, 20) and monoclonal antibodies
(21-24) have been obtained to develop immunoassays. The
most sensitive assay (19) showed a limit of detection of 0.03
ng mL-1 (minimal value), with 50% inhibition of antibody
binding at 0.2 ng mL-1 of chlorpyrifos.
Currently, commercial ELISA-based microtiter plate kits for
detecting residues of chlorpyrifos are available from Strategic
Diagnostics Inc. (Newark, DE) (RaPID assay and EnviroGard)
and EnviroLogix Inc. (Portland, ME). RaPID assay is the most
sensitive (I50 0.3 ng mL-1) but is the least selective, with a high
cross-reactivity to chlorpyrifos-methyl (600%). EnviroLogix kit
is the least sensitive (I50 1.5 ng mL-1) but it is more selective,
since cross-reactivity for chlorpyrifos-methyl is only 6.5%.
Determination of chlorpyrifos in environmental and food
samples by immunoassay is difficult due to its hydrophobicity.
Some authors have reported plastic adsorption of chlorpyrifos,
resulting in low recoveries and inaccurate results (19, 22).
Therefore, a careful use of the equipment (pipets, tips, ELISA
plates) is necessary to minimize analyte losses.
In this work, a novel indirect enzyme-linked immunoassay
for chlorpyrifos has been developed in order to improve both
sensitivity and selectivity. With this aim, a collection of haptens
was synthesized to produce polyclonal antibodies against
chlorpyrifos. Also, the application of the optimized ELISA to
the determination of this compound in olive oil with a simple
and fast sample pretreatment was another goal.
MATERIALS AND METHODS
Reagents. Analytical grade solvents were from Scharlab (Barcelona,
Spain). Pesticide standards used for cross-reactivity studies were
purchased from Sigma-Aldrich Qu´ımica (Madrid, Spain) and Dr.
Ehrenstorfer (Augsburg, Germany). Bovine serum albumin (BSA),
ovalbumin (OVA), complete and incomplete Freund’s adjuvant, o-phen-
ylenediamine (OPD), Tween 20, horseradish peroxidase (HRP), and
peroxidase labeled goat anti-rabbit immunoglobulins (GAR-HRP) were
from Sigma. Chemical reagents for hapten synthesis and protein con-
jugation purposes were from Aldrich (Madrid, Spain). Keyhole limpet
hemocyanin (KLH) was from Pierce (Rockford, IL). Technical quality
chlorpyrifos (98%) was generously provided by Laboratorios Alcotan
(Sevilla, Spain).
Figure 1. Chemical structure of chlorpyrifos and the haptens synthesized
in this work.
To a solution of chlorpyrifos (3.5 g, 10 mmol) in absolute ethanol
(25 mL) was added 25 mL of a solution of KOH (1.42 g, 20 mmol) in
absolute ethanol. After reflux for 1 h, the reaction mixture was filtered
quickly and the solvent evaporated. A solution of NaHCO3 (5%, 25
mL) was added to the white solid obtained, which was extracted with
hexane (3 × 10 mL). The aqueous layer was acidified with phosphoric
acid until the formation of a precipitate. Then, the mixture was extracted
with dichloromethane (3 × 10 mL), dried, and concentrated to give
3,5,6-trichloro-2-pyridinol as a pure, white solid (1.79 g, 90%). 1H NMR
(CDCl3, 300 MHz) δ (ppm): 7.81 (1H, s, CH). 13C NMR (CDCl3, 300
MHz) δ (ppm): 158.2 (COH), 141.5 (C-N), 141.4 (CH), 120.5 (CCl),
117.8 (CCl).
Apparatus. 1H and 13C nuclear magnetic resonance (NMR) spectra
were obtained with a 300 Varian spectrometer (300 MHz, Sunnyvale,
CA).
UV-vis spectra were recorded on a Hewlett-Packard 8452 diode
array spectrophotometer (Palo Alto, CA). Polystyrene 96-well microtiter
plates were from Costar (Cambridge, MA), and the ELISA plate washer
was from Nunc MaxiSorp (Roskilde, Denmark). Well absorbances were
measured in a microtiter plate reader (Wallac, model Victor 1420
multilabel counter, Turku, Finland).
For GC analysis, a 5980 series II Hewlett-Packard automatic sampler
equipped with a DB-1701 capillary column (30 m length × 250 µm
diameter × 0.25 µm film thickness; J & W Scientific, Folsom, CA)
and a flame photometric detector was used.
Five haptens (named C1, C2, C3, C4, C5) have been used in this
work. The structures are shown in Figure 1.
Gel permeation chromatography (GPC) was carried out with an
automated system (GPC VARIO, LCTech, Germany) equipped with a
5-mL loop and a chromatographic column (500 × 25 mm id) filled
with 50 g of BioBeads S-X3-resin (200-400 mesh), with a 32-cm gel
bed length.
The hapten C1, 3-[3,5-dichloro-6-(diethoxythiophosphoryloxy)-
pyridin-2-ylsulfanyl]propionic acid, was prepared following the pro-
cedure of Manclu´s et al. (22).
Haptens C2, 6-[ethoxy-3,5,6-trichloropyridin-2-yloxy)thiophos-
phorylamino]hexanoic acid, and C3, 4-[ethoxy-3,5,6-trichloropyridin-
2-yloxy)thiophosphorylamino]butyric acid, were synthesized following
the procedure described by Cho et al. (20).
Hapten C4, (3-hydroxypropyl)thiophosphoramidic acid O-ethyl ester
O′-(3,5,6-trichloropyridin-2-yl) ester, is a novel chlorpyrifos hapten that
contains a hydroxyl group for protein conjugation. This compound was
obtained following a strategy similar to those applied by Beasley et al.
Hapten Synthesis. In general, the synthesis of chlorpyrifos haptens
has been carried out from its metabolite, 3,5,6-trichloro-2-pyridinol
(TCP), as the starting material. This compound is commercially
available only as standard quality, which implies a high cost for
synthetic purposes. For this reason, the synthesis of TCP was carried
out by hydrolysis of chlorpyrifos, as follows:

9354 J. Agric. Food Chem., Vol. 53, No. 24, 2005
Brun et al.
(25) to diazinon, in two steps. First, thiophosphorochloridic acid O-ethyl
ester O′-(3,5,6-trichloropyridin-2-yl) ester was obtained as follows:
To a stirred mixture of ethyldichlorothiophosphate (1.9 mL, 13.3
mmol) in acetonitrile (24 mL) were added K2CO3 (6.8 g) and 3,5,6-
trichloro-2-pyridinol (2 g, 10 mmol) dissolved in acetonitrile (6 mL).
After stirring for 1 h at room temperature, the mixture was filtered
and the solvent was removed under reduced pressure. The residue was
column chromatographed (silica gel, hexane/diethyl ether 95/5), giving
62 mM sodium phosphate, pH 5.5). After 10 min, the enzymatic reaction
was stopped by adding 2.5 M H2SO4 (50 µL/well) and the absorbance
was read.
The influence of different organic solvents normally used in
extraction procedures was also evaluated. For this purpose, several
percentages of acetone, acetonitrile, ethyl lactate, 2-propanol, methanol,
and methyl sulfoxide in the optimized buffer were tested.
Analysis of Oil Samples. Extra virgin olive oil commercial samples
were collected from representative Spanish producing areas.
Samples were analyzed for chlorpyrifos residues by optimized ELISA
after being fortified and extracted following a previously developed
protocol (29). Briefly, 0.5 mL of the olive oil was mixed with 0.5 mL
of methanol by vortex. The mixture was frozen at -80 °C for 1 h and
then the methanolic extract was collected. Samples were quantified
using standards in olive oil extracted as described.
1
the product as a colorless oil (1.47 g, 43%). H NMR (CDCl3, 300
MHz) δ (ppm): 7.91 (1H, s, CH), 4.57-4.46 (4H, dq, J ) 11.0 and
7.1 Hz, CH2CH3), 1.53-1.48 (6H, dt, J ) 7.1 and 1.1 Hz, CH2CH3).
In a second step, 0.5 g (1.47 mmol) of the previously obtained
compound was dissolved in acetonitrile (11 mL) and the solution cooled
in an ice bath. Then, NaHCO3 (327 mg) and 3-amino-1-propanol (223
mg, 2.97 mmol) were added and the mixture stirred overnight at 4 °C.
Next, the reaction mixture was filtered and the solvent evaporated. The
residue was purified by chromatographic column (hexane/ethyl acetate
Prior to GC analysis, 5 mL of each sample was mixed thoroughly
with 25 mL of ethyl acetate-cyclohexane (1/1, v/v) in 100-mL
Erlenmeyer flasks, and the mixture was stirred at 300 rpm for 24 h at
room temperature. Afterward, 7 mL of supernatant was filtered through
a nylon syringe filter (0.45 µm pore size, 25 mm diameter; Whatman
Inc., NJ). Subsequently, a 5-mL aliquot of the sample extract was
cleaned up by gel permeation chromatography (GPC). The mobile phase
1
7/3) to give a white oil (300 mg, 54%). H NMR (CDCl3, 300 MHz)
δ (ppm): 7.86 (1H, s, CH), 4.40-4.29 (2H, dq, J ) 7.2 and 6.9 Hz,
CH2CH3), 3.83 (2H, t, J ) 5.7 Hz, CH2OH), 3.85-3.70 (1H, m, NH),
3.40-3.25 (2H, m, CH2N), 1.81 (2H, m, CH2), 1.41 (3H, t, J ) 6.9
Hz, CH2CH3). 13C NMR (CDCl3, 300 MHz) δ (ppm): 151.3 (CO),
143.9 (CN), 141.1 (CH), 126.5 (CCl), 121.0 (CCl), 64.3 (CH2OH), 60.4
(CH2CH3), 39.7 (CH2N), 33.0 (CH2), 15.9 (CH3).
Hapten C5, 3,5,6-trichloro-2-pyridyloxyacetic acid (triclopyr), was
generously provided by DowElanco (Indianapolis, IN) and used only
for coating purposes.
Immunoreagents Preparation. For immunization purposes, haptens
were covalently attached through their carboxylic acid moieties to the
lysine groups of BSA and KLH by the active ester method (26).
Additionally, the set of haptens was covalently attached to HRP to
prepare enzyme tracers and to OVA to obtain coating conjugates, both
by the same method.
Hapten C4 was conjugated through its hydroxyl group to BSA, OVA,
and HRP following the method described by Beasley et al. (25).
Finally, immunogens, tracers, and coating conjugates were purified
by gel-exclusion chromatography on D-Salt desalting columns (Pierce,
Rockford, IL) using PBS (10 mM, pH 7.4) for elution. The conjugates
were stored at -20 °C until use.
was ethyl acetate-cyclohexane (1/1, v/v) at a flow rate of 5 mL min-1
.
The first 85 mL (17 min) of the oil extracts was discarded; the following
100 mL was collected as the pesticide fraction (20 min) and reduced
to dryness in a rotary evaporator. Finally, the residue was redissolved
in 1 mL ethyl acetate-cyclohexane (1/1, v/v) and analyzed by GC
with flame photometric detection (FPD). The chromatographic deter-
mination was based on the method described by Jongenotter et al (30),
with modifications.
The GC conditions were as follows: column temperature, 60 °C (1
min), from 60 to 120 °C at 30 °C min-1, from 120 to 220 °C at 5 °C
min-1 (held for 3 min), 15 °C min-1 to 280 °C (held for 24 min);
carrier gas, helium; injection temperature, 250 °C; injection volume, 2
µL with HP 7673 autosampler; injection mode, splitless; detector
temperature, 300 °C.
RESULTS
The immunogens (0.20 mg in 0.5 mL PBS) were suspended in 0.5
mL of Freund’s adjuvant and injected intramuscularly into two fe-
male New Zealand rabbits (I and II). After several boosts, whole blood
was collected and coagulated overnight at 4 °C. Then, serum was
separated by centrifugation and stored at -80 °C. Fourteen sera, from
immunogens BSA-C1, BSA-C2, BSA-C3, BSA-C4, KLH-C2,
KLH-C3, and KLH-C4, were obtained.
To test sera recognition, optimal concentrations of coating conjugates,
serum dilution, and enzyme tracers were chosen by checkboard titration
(27). For this purpose, two assay formats were studied: (a) indirect
(coating conjugate) and (b) direct (antibody-coated).
Hapten Design and Synthesis. To design specific haptens
for a compound, it is desirable to obtain a mimic of its stru-
cture and electronic and hydrophobic properties. Taking this
into account, two types of haptens were synthesized by attaching
the spacer arm on different sites of the structure: the pyridyl
ring (hapten C1) and the thiophosphate moiety (haptens
C2-C4) (Figure 1).
The election of haptens was based on the results obtained by
several researchers (19-24), who have developed sensitive
ELISAs for chlorpyrifos.
ELISA Optimization. Assay optimization was performed with
chlorpyrifos as the competitor analyte following the Tijssen’s protocols
(28). Standards were prepared in distilled water from a stock solution
in methanol. Borosilicate glass tubes were used to minimize chlorpyrifos
loss.
Hapten C1 was prepared by chlorine substitution using
3-mercaptopropionic acid as nucleophile. This structure main-
tains the thiophosphate moiety, having the spacer arm as an
aromatic ring substituent.
Haptens C2 and C3 were synthesized, respectively, by
introduction of 6-aminopropionic acid and 4-aminobutanoic acid
as amide linkage to the thiophosphate ester. This structure has
provided highly specific antibodies for other organophosphorus
compounds (31).
Previous studies carried out by the authors for diazinon and
fenthion (32, 33) showed that haptens with a spacer arm attached
through the phosphate ester and ended with a hydroxyl group
were the most successful for immunizing purposes. Accordingly,
a new hapten (C4) was designed. Briefly, the synthesis involved
the reaction of ethyl dichlorothiophosphate with the sodium salt
of 3,5,6-trichloro-2-pyridinol and the subsequent displacement
of the chlorine by the amino substituent of 3-amino-1-propanol.
Serum Screening. All sera were tested against homologous
and heterologous coating conjugate formats to determine the
Antibodies and tracers working solutions were prepared in buffer
and mixed with an equivalent volume of standards in the plate.
In brief, flat-bottomed polystyrene ELISA plates were coated with
100 µL/well of the appropriate concentration of OVA-triclopyr
conjugate solution in coating buffer (50 mM carbonate-bicarbonate
buffer pH 9.6). The plates were then sealed and incubated overnight at
4 °C. The following day, plates were washed six times with PBS-T
(10 mM phosphate buffer, 137 mM NaCl, 2.7 mM KCl, pH 7.5
containing 0.05% Tween 20). After that, a volume of 50 µL of the
appropriate sera dilution in the tested buffer and 50 µL of standards in
deionized water were added to the coated plates and incubated for 1 h
at room temperature. After washing as earlier, plates were incubated
for 1 h with peroxidase-labeled goat anti-rabbit immunoglobulins
(GAR-HRP) diluted 1:4000 in PBST (100 µL/well). Once washed,
peroxidase activity was determined by adding 100 µL/well of substrate
solution (2 mg mL-1 OPD and 0.012% H2O2 in 25 mM sodium citrate,

Sensitive Enzyme-Linked Immunosorbent Assay for Chlorpyrifos
J. Agric. Food Chem., Vol. 53, No. 24, 2005 9355
Table 1. Titration: Comparison of Coating Conjugates and Antibodiesa
Table 2. I50 Values for the Best Serum-Coating Conjugate
Combinations
serum
OVA−C1
OVA−C2
OVA−C3
OVA−C4
OVA−C5
-
1
serum
coating conjugate
I
50 (ng mL
)
BSA
−
−
−
−
−
−
−
−
C1-I
M
L
L
L
L
L
L
N
L
BSA
BSA
BSA
BSA
BSA
BSA
BSA
C1-II
C2-I
C2-II
C3-I
C3-II
C4-I
C4-II
C2-I
C2-II
C3-I
C3-II
C4-I
C4-II
M
N
M
N
N
N
N
N
N
N
N
N
N
BSA
−
−
−
−
C1-II
OVA
OVA
OVA
OVA
OVA
OVA
OVA
OVA
−C4
−C5
−C5
−C5
−C5
−C5
−C5
−C5
C5
C5
C5
1600
32
H
H
M
M
N
L
H
H
H
M
N
L
M
H
H
H
N
L
H
H
H
H
N
L
M
H
M
M
N
L
H
H
H
H
N
L
M
H
N
M
N
N
M
H
H
L
BSA
BSA
BSA
C2-I
C2-II
C3-II
C2-I
C2-II
C3-I
C3-II
C2-I
C2-II
C3-II
0.04
3
KLH
KLH
KLH
KLH
KLH
KLH
KLH
−
−
−
−
−
−
−
10
7
2
7
7
4
3
KLH
KLH
KLH
KLH
KLH
KLH
−
−
−
−
−
−
BSA
BSA
BSA
−
−
−
N
N
Table 3. Influence of pH, Buffer Concentration, Tween 20, and
Incubation Time on I50 and Assay Bindinga
serum
BSA−C1
BSA−C2
BSA−C3
BSA−C4
BSA−C5
KLH
−
−
−
−
−
−
C2-I
L
M
L
L
N
N
H
H
H
H
N
L
H
H
H
H
N
L
M
M
H
L
N
N
M
H
H
M
N
N
assay Ab
assay Bc
KLH
KLH
KLH
KLH
KLH
C2-II
C3-I
C3-II
C4-I
C4-II
-1
-1
variable
I
50 (ng L
)
I
50 (ng L
)
A0
A0
pH
4
5
6
7.5
8
1348
1798
1348
1809
2023
2472
0.871
1.065
1.000
0.831
0.995
1.029
3.5
0.514
0.709
0.847
0.944
0.969
1.009
5.8
19.8
39.6
59.3
79.1
a L (low), M (medium), and H (high) correspond to the dilution factor applied to
the sera for coating conjugate. Coating conjugate concentration ranges from 0.001
-
1
1
9
to 1.0 mg L to obtain an absorbance signal between 0.5 and 1.2. L <
/
10 000, M
1
1
1
PBS (mM)
0
5
10
20
40
80
)
/
to
/
30 000, and H >
/30 000. N means not detected. I and II are sera from
10 000
19551
12412
10943
1348
974
0.783
0.869
0.953
1.000
0.864
0.834
1137.6
741.9
79.1
19.8
9.9
1.120
0.870
0.936
0.847
0.647
0.468
different rabbits.
titers. First, the behavior of the sera using an antibody-coated
format with all HRP conjugates was investigated. None of the
sera showed enough titer to carry out competitive assays.
All haptens were conjugated to OVA and BSA and used as
coating conjugates. The results of the screening of the antibodies
in indirect immunoassay format are presented in Table 1.
Sera from hapten C1, the only hapten with the spacer arm
attached to the aromatic ring, presented low titer when hapten
C2, C3, or C4 was used for coating purposes.
Haptens C2 and C3, which differ only in the length of the
spacer arm, rendered sera with higher titers than the other
haptens, especially C2, which has the larger spacer arm.
Nevertheless, sera obtained with hapten C4 showed lower titers,
although its structure is similar to that of C3, differing only in
the attaching group to proteins (-OH).
824
4.7
Tween 20 (%)
0
0.005
0.01
0.05
0.1
0.5
time (min)
15
75688
6404
1742
1381
1348
365
0.744
0.928
0.841
1.003
1.000
0.989
10.8
17.9
21.8
18.1
19.8
146.3
1.217
1.144
1.059
0.908
0.848
0.562
202
270
271
365
0.649
0.802
0.838
0.989
4.3
6.9
8.6
0.849
1.023
1.123
1.217
30
45
60
10.8
a RSD ranges from 1% to 8%. b Assay A, pair KLH
−
C3-I/OVA
−
C5 (1:6000/1
-
-
mg L 1). c Assay B, pair BSA
−C2-II/OVA
−
C5 (1:4000/0.01 mg L 1).
For coating, the use of haptens without the thiophosphate
group and with spacer attachment by O-alkylation has provided
the best results in other works. Also, studies about spacer arm
length indicated that the shorter the arm, the higher the
sensitivity (32, 33). Applying this strategy to chlorpyrifos, hapten
C5 was tested as coating hapten, to introduce a major degree
of heterology. As can be seen in Table 1, OVA-C5 conjugate
was highly recognized by sera obtained from haptens C2 and
C3.
All the combinations of serum/coating conjugates that showed
specific recognition were used to carry out competitive assays
in order to set up the most sensitive assay for chlorpyrifos.
Results for more sensitive combinations are given in Table 2.
The pairs KLH-C3-I/OVA-C5 (1:6000/1 mg L-1) (assay A)
and BSA-C2-II/OVA-C5 (1:4000/0.01 mg L-1) (assay B)
were selected for immunoassay optimization purposes, due to
its wide dynamic range and its low I50 value, respectively.
ELISA Optimization. The effect of pH, ionic strength,
surfactant concentration, and incubation time on assay perfor-
mances (A0 and I50) was studied at room temperature for the
indirect ELISA format. Results are shown in Table 3.
First, the effect of pH between 4.0 and 9.0, using PBS-T (20
mM 0.1% v/v Tween 20) and 1 h of incubation time was tested.
I50 values and maximum signal (A0) do not change significantly
in this pH range for the immunoassay A and both parameters
increase when pH does for assay B. The best combinations of
I50/signal were obtained at pH 6.0. So it was selected to continue
the assays optimization.
To estimate the influence of salt concentration, PBS-T
solutions at 0, 5, 10, 20, 40, and 80 mM, all containing 0.1%
v/v Tween 20 at the optimized pH, were tested. In all cases, I50
values decreased gradually as concentration increased, as had
been previously observed for other organophosphorus immu-
noassays, probably due to similar biochemical interactions
between analyte and antibody. Also, the maximum signal
decreased for both immunoassays when the concentration is
higher than 20 mM. In this study, a concentration of 20 mM
PBS was selected as a compromise between maximum A0 signal
and minimum I50.
Also, the effect of surfactant concentration (0, 0.005, 0.01,
0.05, 0.1, and 0.5% Tween, v/v) on immunoassay performance

9356 J. Agric. Food Chem., Vol. 53, No. 24, 2005
Brun et al.
Figure 2. Calibration curve for the optimized chlorpyrifos immunoassays in different media. Mean values
± standard deviation (n ) 3).
was studied, using the optimal pH and PBS concentration. For
assay A, lower I50 values were observed when Tween 20
concentration increased. For this reason, 0.5% Tween 20 was
selected.
On the other hand, the lowest I50 value was obtained for assay
B when no surfactant was added to the buffer, and this condition
was selected to carry out the assay.
showed a lower CR (26%) for this compound, probably because
the hapten used for immunization maintains both ethoxyl groups
in the phosphate ester. Chlorpyrifos-methyl is not a troublesome
interferent, as it is present in many commercial chlorpyrifos
formulations (18).
For chlorpyrifos-oxon, the cross-reactivity values were higher
than those given by other authors, but similar (30%) to that
reported by Hill et al. (19).
On the other hand, the cross-reactivity to TCP is negligible.
Since this is the main chlorpyrifos metabolite in water, it is
possible to determine chlorpyrifos in aqueous media without
TCP interference.
The optimized ELISAs showed interference with bromo-
phos-methyl, but slight cross-reactivity with bromophosethyl.
Bromophos-methyl had lower CR (4.6%) for the assay de-
scribed by Cho et al. (20), and no data were reported by other
authors.
A cross-reactivity of 15% for fenchlorphos was displayed for
assay B, but it was negligible with assay A. Reported immu-
noassays for chlorpyrifos showed variable cross-reactivity for
this compound (6 and 75%) (19, 21).
It is remarkable that 15 of the 18 compounds tested did not
have appreciable cross-reactivity for both chlorpyrifos immu-
noassays developed in this work.
Tolerance to Organic Solvents. The effect of different
organic solvents, which are necessary for pesticide extraction
in complex matrixes, was studied in the optimized immunoas-
says. As is shown in Table 5, the assay A tolerated the tested
PBS-organic solvents mixtures worse than assay B. Methanol
was one of the best tolerated solvents by assay A (up to 20%).
Acetonitrile and acetone were well-tolerated up to 10%,
2-propanol and methyl sulfoxide up to 4%, but ethyl lactate
only up to 2%. In some cases, 2-propanol at 1% and 2% showed
better I50 than pure PBS-T.
Finally, the influence of the incubation time on the competi-
tion step (15, 30, 45, and 60 min) was investigated. I50 and A0
values increased gradually as time increased. So, competition
times of 30 min for assay B and 45 min for A were selected in
order to obtain sensitive assays with an appropriate signal, in
the minimum time. Shorter times can be also used, but the assay
reproducibility is lower.
After optimization, the assay A exhibited an I50 of 271 ng
L-1, a working range between 4 and 16 474 ng L-1, and a
LOD of 0.3 ng L-1. Although assay A has an I50 similar to
the previously best reported chlorpyrifos’ ELISAs (19, 23), the
limit of detection is considerably low and the competitive
curve shows a wide dynamic range. The immunoassay B
exhibits an I50 of 7 ng L-1, a working range between 0.4 and
302 ng L-1, and a LOD of 0.07 ng L-1. This is, to our
knowledge, the most sensitive immunoassay reported for
chlorpyrifos. Figure 2 illustrates the competition curve of each
assay, showing a good repeatability for results from assay B
(average RSD ) 3%), but a worse one for assay A (average
RSD ) 4%) (dotted lines).
Cross-Reactivity Studies. Assay selectivity was evaluated
using a set of organophosphorus insecticides and metabolitess
because of their similar structure to chlorpyrifossand several
nonchemically related pesticides, due to their widespread
agricultural and domestic use. I50 and CR data for each
compound are given in Table 4.
In both ELISA’s, there were interferences with chlorpyrifos-
methyl, as it had been previously reported in other works (19,
20, 23). Only the immunoassay described by Lawruk et al. (24)
Assay B tolerated well all tested percentages of 2-propanol.
In fact, the assay is more sensitive if this solvent is used

Sensitive Enzyme-Linked Immunosorbent Assay for Chlorpyrifos
J. Agric. Food Chem., Vol. 53, No. 24, 2005 9357
Table 4. Cross-Reactivity of Chlorpyrifos-Related Compounds, Metabolites, and Nonrelated Pesticidesa
-
-
a Assay A, pair KLH
pH 6. Average RSD
−
C3-I/OVA
−
C5 (1:6000/1 mg L 1), PBS 20 mM, 0.5% v/v Tween 20, pH 6. Assay B, pair BSA
−
C2-II/OVA
−
C5 (1:4000/0.01 mg L 1), PBS 20 mM,
)
5%.
compared with PBS alone, although the signal decreases at
percentages higher than 4% of 2-propanol. Up to 10% of methyl
sulfoxide can be used as well with the same performances and
methanol, acetonitrile, and acetone up to 4%. Ethyl lactate was
the least favorable solvent (tolerated up to 2%). As can be seen,
even under the worst conditions the I50 values are good,
maintaining the ng mL-1 sensitivity level.
Methanol and 2-propanol were the best tolerated solvents for
the developed immunoassays. 2-Propanol partially dissolves the
olive oil, making difficult the subsequent extraction and analysis.
Therefore, methanol was the selected solvent for extraction
purposes. Moreover, previous studies carried out by the authors
on pesticide extraction in olive oil samples led to a simple and
effective protocol using methanol (29).

9358 J. Agric. Food Chem., Vol. 53, No. 24, 2005
Brun et al.
Table 5. Effect of Organic Solvents on Chlorpyrifos Immunoassays
Table 6. Chlorpyrifos Determination by ELISA in Spiked Extra Virgin
Performancea
Olive Oil Samplesa
assay Aa
assay Bb
assay A
[found]
assay B
[found]
-1
-1
sample
[spiked]
R
R
medium
buffer
methanol
1
2
4
10
20
acetonitrile
1
2
4
10
20
I50 (ng L
271
)
I50 (ng L
7
)
A0
A0
1
2
3
4
5
6
7
8
1000
800
500
350
200
100
95
50
20
10
1156
918
488
409
200
91
98
57
26
7
±
±
±
±
±
±
±
±
±
±
159
249
54
60
20
23
14
4
116
115
98
117
100
91
103
114
130
70
1027
688
481
367
203
119
76
45
19
10
±
±
±
±
±
±
±
±
±
±
18
36
29
21
6
3
4
3
2
1
103
86
96
105
102
119
80
90
95
100
98
0.838
1.023
298
244
352
325
678
0.846
0.612
0.797
0.817
0.835
7
7
7
0.998
0.972
0.945
0.950
0.886
81
6500
9
10
mean
2
1
379
285
222
214
nc
0.955
0.916
0.857
0.717
0.586
3
6
11
ncc
nc
1.119
1.099
1.037
2.161
0.323
103
a [Spiked] and [found] values are expressed in ng mL
± standard deviation (n
-
1
acetone
1
2
4
10
20
)
3). R, recovery, expressed in % respect to the spiking levels.
339
298
314
599
886
0.885
0.711
0.798
0.768
0.519
4
4
9
3806
1.052
1.032
0.930
0.717
0.222
Table 7. Comparison of Chlorpyrifos Determination by ELISA and GC
Methods in Spiked Olive Oil Samplesa
19956
assay A
[found]
240 42
assay B
[found]
224 31
488
GC
[found]
2-propanol
1
2
4
10
20
methyl sulfoxide
1
2
4
10
20
ethyl lactate
1
2
4
10
20
173
152
369
139901
510044
0.859
0.845
0.760
0.585
0.184
5
6
3
2
2
1.043
0.977
1.026
0.898
0.759
sample [spiked]
R
R
R
1
2
200
500
±
120
101
±
112
98
248
475
114 1035
106 205
±
±
±
±
±
±
38
45
124
95
507
1247
208
1145
496
±
±
±
±
±
43
39
11
±
±
±
±
±
54
39
22
3
4
1000
200
125 1137
104 212
154 104
56 103
153 127
5
6
1000
500
159 115 1143
38
136 114 1270
270
291
428
8282
339834
0.867
0.698
0.781
0.714
0.526
4
8
7
9
1.056
0.993
0.957
0.852
0.567
99
111
497
42
99
107
528
52
106
110
mean
a [Spiked] and [found] values are expressed in ng mL
3). R, recovery, expressed in % respect to the spiking level.
± standard deviation (n
-
1
1235
)
325
407
7046
nc
0.863
0.729
0.664
0.325
1.279
21
58
3134
nc
nc
1.048
0.920
0.736
0.253
0.889
Finally, six oils were analyzed as blind samples by immu-
noassays and gas chromatography methods. The obtained results
(Table 7) showed good correlations with the reference
method: for assay A, y ) 0.933x + 29.376 ng mL-1, r ) 0.969;
for assay B, y ) 1.006x + 6.469 ng mL-1, r ) 0.985.
The results show that the developed ELISAs are suffi-
cient to quantify chlorpyrifos levels below the established
MRL (50 µg kg-1) in olive fruit for olive oil production by
the Spanish legislation. Also, it is worth mentioning the
high chlorpyrifos extraction and quantification throughput
using both optimized methodologies. Our procedure is com-
petitive, reducing considerably the cost and analysis time
compared with the reference chromatographic method. Al-
though immunoassay has demonstrated to be a valuable tool
for the screening of a high number of samples, confirmatory
analysis by chromatographic techniques should be carried out,
as chromatography offers automation, robustness, and high
sensibility.
nc
a Assay A, solvent percentage in PBS 20 mM, 0.5% v/v Tween 20, pH 6.
b Assay B, solvent percentage in PBS 20 mM, pH 6. c nc means no competitive
curve.
The features of the immunoassays at 2% methanol (final
concentration in ELISA plate) were an I50 of 244 ng L-1, a
working range between 74 and 2688 ng L-1, and a LOD of
22 ng L-1 for assay A (Figure 2). An I50 of 7 ng L-1, a
working range between 1.4 and 487 ng L-1, and a LOD of
0.4 ng L-1 were reached for assay B. Under these conditions,
the assays can be applied for chlorpyrifos determination with
similar sensitivity to the calibration curve obtained without
methanol.
Analysis of Olive Oil Samples. Ten extra virgin olive oil
samples (free of chlorpyrifos, as determined by GC) were
analyzed through the developed assays. Samples were spiked
at different chlorpyrifos levels and extracted as described in
Materials and Methods. The methanolic extracts were diluted
1:25 in distilled water (2% methanol in plate) and quantified
by ELISA using sera diluted in the optimized buffer.
Poor chlorpyrifos recoveries were achieved using standard
curves in 2% methanol due to the matrix effect (Figure 2), as
was also observed in previous studies (29). To normalize for
matrix effects, the standard curves were made using spiked olive
oil and extracted in the same way as the samples. As can be
seen in Table 6, the recovery values obtained using standard
curves in oil matrix were satisfactory, between 70% and 130%
for assay A and between 80% and 119% for assay B. Assay B
exhibited better precision than assay A.
DISCUSSION
A new hapten for chlorpyrifos has been synthesized, although
the best sera were obtained with previously described haptens.
Nevertheless, important improvement in the sensitivity compared
with reported chlorpyrifos immunoassays has been achieved
using a different coating conjugate, lacking the thiophosphate
ester, due to heterology effects.
ELISAs have been developed to determine chlorpyrifos and
applied to the screening of olive oil samples. The methodology
offers strong advantages, such as simple extraction, rapidity,
sensitivity, and good reproducibility, despite the characteristics
of the matrix. These immunochemical techniques offer advan-

Sensitive Enzyme-Linked Immunosorbent Assay for Chlorpyrifos
J. Agric. Food Chem., Vol. 53, No. 24, 2005 9359
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compound, chlorpyrifos.
One of the developed chlorpyrifos immunoassays (assay B)
is, to our knowledge, the most sensitive, exhibiting also a good
tolerance to organic solvents, which makes it very valuable for
its application in complex matrixes.
ACKNOWLEDGMENT
(17) Lo´pez-Rolda´n, P.; Lo´pez de Alda, M. J.; Barcelo´, D. Simulta-
neous determination of selected endocrine disrupters (pesticides,
phenols and phthalates) in water by in-field solid-phase extraction
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The authors thank Dr. J. A. Gabaldo´n for valuable GC analysis
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providing chlorpyrifos.
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JF0516641