Preparation of antibodies and development of an enzyme-linked immunosorbent assay for determination of dealkylated hydroxytriazines

Article abstract of DOI:10.1021/jf025640v

The development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for dealkylated hydroxytriazines is reported here for the first time. The assay uses polyclonal antibodies raised against N-(4-amine-6-hydroxy-[1,3,5]triazin-2-yl)-4-aminobutanoic acid (hapten 2g) conjugated to keyhole limpet hemocyanin by the active ester method. The immunizing hapten was synthesized by first introducing the amino group to the triazine ring in a protected form in order to increase its solubility in organic media. Subsequent steps consisted of reacting this compound with an appropriate spacer arm, followed by removal of the protecting group in acidic media. The resulting assay uses a homologous competitor hapten coupled to conalbumin by the mixed anhydride method. Coating antigens prepared using a homologous covalent coupling procedure failed to produce competitive immunoassays. The assay tolerates media with high ionic strength (up to 70 mS cm^-1) and basic pH values (7.5-9.5 units). Under the optimized conditions, this ELISA is specific for dealkylated hydroxytriazines, reaching suitable limits of detection.

Full text of DOI:10.1021/jf025640v

156 J. Agric. Food Chem. 2003, 51, 156−164
Preparation of Antibodies and Development of an
Enzyme-Linked Immunosorbent Assay for Determination of
Dealkylated Hydroxytriazines
NURIA SANVICENS,† VALERIE PICHON,‡ MARIE-CLAIRE HENNION,‡ AND
M.-PILAR MARCO*,†
Department of Biological Organic Chemistry, IIQAB-CSIC, Jordi Girona, 18-26,
08034-Barcelona, Spain, and Laboratoire de Environment et Chimie Analytique,
Ecole Supe´rieure de Physique et de Chimie Industrielles (ESPCI) de Paris, 10, rue Vauquelin,
75231 Paris cedex 05, France
The development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for
dealkylated hydroxytriazines is reported here for the first time. The assay uses polyclonal antibodies
raised against N-(4-amine-6-hydroxy-[1,3,5]triazin-2-yl)-4-aminobutanoic acid (hapten 2g) conjugated
to keyhole limpet hemocyanin by the active ester method. The immunizing hapten was synthesized
by first introducing the amino group to the triazine ring in a protected form in order to increase its
solubility in organic media. Subsequent steps consisted of reacting this compound with an appropriate
spacer arm, followed by removal of the protecting group in acidic media. The resulting assay uses a
homologous competitor hapten coupled to conalbumin by the mixed anhydride method. Coating
antigens prepared using a homologous covalent coupling procedure failed to produce competitive
immunoassays. The assay tolerates media with high ionic strength (up to 70 mS cm-1) and basic pH
values (7.5-9.5 units). Under the optimized conditions, this ELISA is specific for dealkylated
hydroxytriazines, reaching suitable limits of detection.
KEYWORDS: s-Triazines; hydroxylated atrazine degradation products; DEHA; DIHA; immunoassay;
hapten design; hapten heterology
INTRODUCTION
cesses, such as hydrolytic reactions, lead to the formation of
the so-called hydroxylated atrazine degradation products
(HADPs): hydroxyatrazine (HA), deethylhydroxyatrazine
(DEHA), deisopropylhydroxyatrazine (DIHA), and deethylde-
isopropylhydroxyatrazine (DEDIHA). Other important triazine
herbicides, such as propazine, simazine, and tert-butylazine, may
lead to the same kind of metabolites (see Figure 1). It is
important to notice that the half-life of HADP is higher than
those of other s-triazines (in soil: atrazine 14-50 days, HADP
32-162 days) (16), creating a risk for a continuous increase of
the concentration of these metabolites in the environment, the
impact of which in the ecosystem is still unknown (17).
Atrazine and other related s-triazines have been widely used
as selective herbicides for the control of annual grasses and
broad-leaf weeds. Triazine residues have been found in many
compartments of the environment such as sediments and surface,
well, and drinking water due to their extensive use and their
persistence in the environment (1-5). The use of triazines has
been limited in several countries, and their levels in natural
waters are frequently controlled (6-12). However, all the
restrictions adopted in the use of these herbicides have not
solved the problem of the degradation products found in
groundwater and surface water.
In the past decade, most of the studies performed to establish
the impact of these metabolites in the environment have been
focused on the dealkylated degradation products such as DIA,
DEA, and DEDIA. The presence of these compounds in lake
watersheds (18), groundwater (19, 20), and rainfall (1, 21) at
concentrations around 1-5 µg L-1 has been reported. However,
only few studies have been reported regarding HADPs. While
these compounds have been rarely detected in groundwater,
concentrations from 2 to 6 µg L-1 have been reported in surface
water (22, 23). For example, studies performed in watersheds
of northeastern Missouri showed that levels of HA were
generally higher than or equal to those of atrazine or DEA (24).
Triazine herbicides might be degraded by photochemical,
chemical, and/or biochemical processes (13). Hence, several
microorganisms can dealkylate atrazine to produce deethyl-
atrazine (DEA), deisopropylatrazine (DIA), and deethyldeiso-
propylatrazine (DEDIA) (14), although it has been postulated
that these metabolites can also be formed by photochemical
catalysis (1, 15). Moreover, nonbiological detoxification pro-
* Corresponding author (phone +34 93 4006171, fax +34 93 2045904,
E-mail mpmqob@iiqab.csic.es).
† Department of Biological Organic Chemistry, IIQAB-CSIC.
‡ Laboratoire de Environment et Chimie Analytique, ESPCI.
10.1021/jf025640v CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/22/2002

Determination of Dealkylated Hydroxytriazines
J. Agric. Food Chem., Vol. 51, No. 1, 2003 157
Table 1. Chemical Structure of the Atrazine Haptens Used in This
Studya
triazine
R
1
R
2
R
3
2a
2b
2c
2d
2e
2f
2g
4a
4b
4c
4d
4e
NHCH CH
NH(CH ) COOH
Cl
Cl
Cl
Cl
Cl
Cl
OH
2
3
2 3
NHCH(CH )
NH(CH ) COOH
3 2
2 3
NHCH CH
NH(CH ) COOH
2
3
2 5
NHCH(CH )
NH(CH ) COOH
3 2
2 5
NHC(CH )
NH(CH ) COOH
3 3
2 3
NHCH(CH −CH )
NH(CH ) COOH
2
2
2 3
NH
NH(CH ) COOH
2
2 3
NHCH CH
NHCH CH
S(CH ) COOH
S(CH ) COOH
S(CH ) COOH
SCH
2
3
2
3
2 2
NHCH(CH )
NHCH(CH )
3 2
3 2
2 2
NHC(CH )
NHCH(CH −CH )
3 3
2
2
2 2
NHC(CH )
NH(CH ) COOH
3 3
2 3
3
NHCH(CH −CH )
NH(CH ) COOH
SCH
2
2
2 3
3
Figure 1. Chemical structures of the most important s-triazine herbicides
(atrazine, tert-butylazine, propazine, and simazine) and their dealkylated
hydroxy metabolites. The chemical structure of hapten 2g is also shown.
a The preparation of the haptens 2a−f and 4a−e has already been reported
(46−48). The synthesis of 2g, used as an immunizing and coating antigen, is
reported here.
Moreover, it has been shown that HADPs constitute the most
important fraction of the atrazine residues found in soil (16,
25) The higher pKa values of these metabolites (HADPs pKa )
4-5; atrazine pKa ) 2) may allow them to establish mixed-
mode binding mechanisms based on hydrophobic and cationic-
exchange interactions with soil components, while atrazine and
its chlorinated metabolites are limited to hydrophobic interac-
tions (26).
a Varian Unity-300 (Varian Inc., Palo Alto, CA) spectrometer (300
MHz for 1H and 75 MHz for 13C) or a Gemini 200 apparatus (199.975
MHz for 1H and 50.289 for 13C). Infrared (IR) spectra were measured
on a Bomen MB 120 FTIR spectrophotometer (Hartmann & Braun,
Que´bec, Canada). Elemental analyses were performed by Microanalysis
Service of the IIQAB-CSIC in Barcelona. Gas chromatography-mass
spectrometry (GC-MS) was performed on an MD-800 capillary gas
chromatograph with an MS quadrupole detector (Fison Instruments,
VG, Manchester, UK), and the data are reported as m/z (relative
intensity). The ion-source temperature was set at 200 °C; a 15-m ×
0.25-mm-i.d. × 0.15-µm-film thickness DB-225 fused capillary column
(J&W, Folsom, CA) was used; He was the carrier gas employed at 1
mL/min. GC conditions were as follows: temperature program, 70-
80 °C (10 °C/min), 80 °C (5 min), 80-300 °C (10 °C/min); injector
temperature, 250°C.
Environmental monitoring of these metabolites is based on
chromatographic techniques such as high-performance liquid
chromatography with ultraviolet detection (HPLC/UV) or gas
chromatography with mass spectrometry identification (GC/MS)
after organic solvent extraction followed by purification/
concentration steps (7, 27-29). Due to their high polarity and
water solubility, an important drawback of these procedures is
the low efficiency of the procedures for extraction from aqueous
samples (water solubility data: atrazine, 0.15 mM; HA, 0.24
mM; DEA, 2 mM; DIA, 1.20 mM; DEDIA, DEHA, and DIHA,
>2 mM) (30-32). In this context, one of the advantages of the
immunochemical techniques is that they often provide the
necessary detection limits and specificity to directly analyze very
low concentrations of the target analyte in water samples (33-
37). Moreover, their reliability, low cost, speed of analysis, and
easy-to-use features are well known (33, 34, 38-40). The
preparation of antibodies and the development of ELISAs for
DEA, DIA, and DEDIA (41, 42), and also for HA (43-45),
have been described. However, despite the above-mentioned
analytical difficulties, to our knowledge no immunoassay is
available or has been reported to date for the analysis of DEHA,
DIHA, and DEDIHA. In this paper we describe the preparation
of an immunizing hapten and the production of polyclonal
antibodies against dealkylated hydroxytriazines. These antibod-
ies have been used to develop an immunoassay to determine
dealkylated hydroxytriazines.
Synthesis of the Haptens. Most of the chemical reagents needed
for the synthesis of the haptens were obtained from Aldrich Chemical
Co. (Milwaukee, WI). The preparation of the haptens 2a-f and 4a-f
has already been described (46-48) (see Table 1 for chemical
structures). The synthesis of the hapten 2g (6) is described below.
(4,6-Dichloro-[1,3,5]triazin-2-yl)tritylamine (3). A solution of cya-
nuric chloride 1 (1.00 g, 5.4 mmol) in anhydrous Et2O (50 mL) in a
round-bottom flask provided with a CaCl2 tube was cooled to -20 °C.
Subsequently, a solution of tritylamine 2 (1.46 g, 5.6 mmol) and N,N-
diisopropylethylamine (DIEA, 0.98 mL, 5.6 mmol) in anhydrous Et2O
(30 mL) was added dropwise over 1 h. The temperature of the mixture
was then allowed to reach room temperature, and the precipitation of
a white solid corresponding to the DIEA chlorohydrate was observed.
The mixture was stirred for 24 h at room temperature until the reaction
was completed according to the TLC analysis. The mixture was filtered
and washed with aqueous 1 N HCl (50 mL), 10% NaHCO3 (50 mL),
and saturated NaCl (2 × 50 mL). The solution was dried with anhydrous
MgSO4, filtered, and evaporated to dryness under reduced pressure.
The white solid obtained was then dissolved in Et2O and purified by
“flash” chromatography using silica gel as stationary phase and hexane/
AcOEt 4:1 as mobile phase to isolate the triazine 3 as a white powder
1
(1.76 g, 80% yield). H NMR (300 MHz, CDCl3): δ (ppm) 7.08 (bs,
1H, NH), 7.27 (m, 15Har). 13C NMR (75 MHz, CDCl3): δ (ppm) 71.78
(CHN), 127.42 (C-4), 128.10 (C-2, C-6), 128.64 (C-3, C-5), 143.09
(C-1), 165.51 (CN3), 169.43 (CN2Cl), 169.88 (CN2Cl). EM: m/z
EXPERIMENTAL SECTION
+
General Methods and Instruments. Thin-layer chromatography
(TLC) was performed on 0.25-mm precoated silica gel 60 F254
aluminum sheets (Merck, Darmstadt, Germany), and unless otherwise
indicated, the mobile phase employed was THF/AcOEt/hexane 2:13:
35. Anhydrous solvents used were distilled immediately before the
reaction in the presence of Na and under Ar atmosphere (benzophenone
was added as indicator). 1H and 13C NMR spectra were obtained with
(relative intensity) 406 (M+, 25), 329 (M+ - C6H5, 31), 243 (C19H15
,
39), 165 (C13H9+, 100), 104 (C7H6N+, 63), 77 (C6H5+, 47). IR (KBr,
cm-1): ν 3404 (NH st), 1546 (CdN st), 1512 (CArN st), 700 (ArC-H
δ opp). Melting point: 194-196 °C. Anal. Calcd for C22H16N4Cl: C,
64.88; H, 3.96; N, 13.75. Found: C, 64.93; H, 4.10; N, 13.53.
N-(6-Chloro-4-tritylamine-[1,3,5]triazin-2-yl)-4-aminobutanoic acid
(5). In a round-bottom flask provided with a Dimroth refrigerant and

158 J. Agric. Food Chem., Vol. 51, No. 1, 2003
Sanvicens et al.
a CaCl2 tube, a mixture of 3 (1.95 g, 4.8 mmol), 4-aminobutanoic acid
4 (0.54 g, 5.3 mmol), and DIEA (1.75 mL 10.1 mmol) were dissolved
in absolute ethanol (100 mL). After 4 h at 78 °C, the precipitation of
a white solid corresponding to the DIEA chlorohydrate was observed,
together with the complete disappearance of the starting material, as
evidenced by TLC. The mixture was filtered under vacuum and the
solvent evaporated under reduced pressure. The resulting white solid
was then dissolved in a 10% NaHCO3 aqueous solution (200 mL) and
washed with CH2Cl2 (2 × 40 mL). Subsequently, the aqueous layer
was acidified to pH 1 with 0.1 N HCl to precipitate the acid 5. The
white solid, separated by vacuum filtration, was washed with H2O to
remove the resting salts (5 × 20 mL) and freeze-dried to rend the
triazine 5 as a white powder (2.19 g, 98% yield). 1H NMR (300 MHz,
CD3OD): δ (ppm) 1.23 (m, 2H, J ) 5.1 Hz, CH2), 1.81 (t, 2H, J )
7.2 Hz, CH2COO), 2.53 (t, 2H, J ) 7.2 Hz, CH2N), 7.17 (m, 15Har).
13C NMR (75 MHz, DMSO-d6): δ (ppm) 24.26 (CH2), 31.85 (CH2-
COO), 47.76 (CH2N), 69.73 (CHN), 126.21 (C-4), 127.30 (C-2, C-6),
128.42 (C-3, C-5), 144.51 (C-1), 164.13 (CN3), 164.97 (CN3), 167.13
(CN2Cl), 175.21 (CO). IR (KBr, cm-1): ν 3427 (NH st), 3269 (NH
st), 1712 (CdO), 1577 (CdN st), 1537 (CArN st), 700 (ArC-H δ opp).
N-(4-Amine-6-hydroxy-[1,3,5]triazin-2-yl)-4-aminobutanoic Acid (6)
(Hapten 2g). A mixture of trifluoroacetic acid (TFA), H2O, and CH2-
Cl2 (45:45:10, 20 mL) was added to the triazine 5 (1.17 g, 2.5 mmol)
in a round-bottom flask. The resulting mixture was heated to reflux
for 2 h until the complete disappearance of the starting material was
observed by 1H NMR. The solvent was evaporated under reduced
pressure to dryness, and the resulting beige solid was washed with
n-pentane (6 × 10 mL) and dried in the presence of P2O5 to obtain
hapten 6 (0.37 g, 70% yield) as a pale beige solid. 1H NMR (300 MHz,
DMSO-d6): δ (ppm) 1.72 (m, 2H, J ) 6.9 Hz, CH2), 2.24 (t, 2H, J )
7.5, CH2COO), 3.25 (bs, 2H, CH2N). The elemental analysis showed
the absence of chlorine atoms in the molecule.
Immunochemistry. Chemicals. Atrazine and simazine, used as stan-
dards for cross-reactivity studies, were purchased though Riedel de Ha¨en
AG (Seelze-Hannover, Germany); desmentryne, 2-hydroxysimazine,
2-hydroxypropazine, DEA, DIA, DEHA, DIHA, DEDIHA, and 2-hy-
droxyatrazine were obtained from Dr. Ehrenstorfer (Ausburg, Germany);
prometryne was purchased from PolyScience Corp. (Niles, IL); finally,
irgarol 1051 was prepared in our group by M. Carmen Este´vez.
Buffers and Solutions. PBS is 0.01 M phosphate buffer, 0.8% saline
solution, and unless otherwise indicated the pH is 7.5. PBST is PBS
with 0.05% Tween 20. PBST-I is 0.05 M PBS, pH 7.5, with 0.1%
Tween 20. Borate buffer is 0.2 M boric acid-sodium borate, pH 8.7.
Coating buffer is 0.05 M carbonate-bicarbonate buffer, pH 9.6. Citrate
buffer is a 0.04 M solution of sodium citrate, pH 5.5. The substrate
solution contains 0.01% tetramethylbenzidine (TMB) and 0.004% H2O2
in citrate buffer. Enzymatic reactions were stopped by adding 4 N H2-
SO4.
room temperature, the activated hapten was added dropwise to a solution
of the protein (BSA, CONA, or OVA, 10 mg) in 0.2 M borate buffer
(1.8 mL).
Polyclonal Antisera. Rabbits 71, 72, and 73 (female New Zealand
white rabbits), weighing 1-2 kg, were immunized with 2g-KLH (EA)
according to the immunization protocol previously reported (46).
Evolution of the antibody titer was assessed by measuring the binding
of serial dilutions of the different antisera to microtiter plates coated
with 2g-BSA (EA). After an acceptable antibody titer was observed,
the animals were exsanguinated, and the blood was collected on
vacutainer tubes provided with a serum separation gel. Antiserum (As)
was obtained by centrifugation and stored at -40 °C in the presence
of 0.02% NaN3. Working aliquots were stored at 4 °C.
Instrumentation. The matrix-assisted laser desorption/ionization time-
of-flight mass spectrometer (MALDI-TOF-MS) used for analyzing the
protein conjugates was a Perspective BioSpectrometry Workstation
provided with the software Voyager-DE-RP (version 4.03), developed
by Perspective Biosystems Inc. (Framingham, MA), and Grams/386
(for Microsoft Windows, version 3.04, level III), developed by Galactic
Industries Corp. (Salem, NH). The pH and the conductivity of all buffers
and solutions were measured with a pH meter (pH 540 GLP) and a
conductimeter (LF 340), respectively (both from WTW, Weilheim,
Germany). Polystyrene microtiter plates were purchased from Nunc
(Maxisorp, Roskilde, Denmark). The vacutainer blood collection set
was acquired from Becton Dickinson (Meylan Ce´dex, France). Washing
steps were carried out using an SLY96 PW microplate washer (SLT
Labinstruments GmbH, Salzburg, Austria). Absorbances were read
using a Spectramax Plus microplate reader (Molecular Devices, Sun-
nyvale, CA) at a single wavelength of 450 nm. The competitive curves
were analyzed with a four-parameter logistic equation using the software
GraphPad Prism (GraphPad Software Inc., San Diego, CA). Unless
otherwise indicated, data presented correspond to the average of at least
two well replicates. The pKa and log P theoretical calculations were
carried out with the program ACD/log P and ACD/pKa (Advanced
Chemistry Development, Inc., Toronto, Canada) at the University of
Lund (Sweden).
Hapten Density Analysis. Hapten densities were determined by
MALDI-TOF-MS by comparing the molecular weight of the standard
BSA and those of the conjugates. MALDI spectra were obtained by
mixing 1 µL of the matrix [(E)-3,5-dimethoxy-4-hydroxycinnamic acid,
10 mg mL-1, in CH3CN/H2O 70:30, 0.1% TFA) and 1 µL of a solution
of the conjugates or proteins (5 mg mL-1 in CH3CN/H2O 70:30, 0.1%
TFA).
ELISA Development and Evaluation. General Protocol. The plates
were coated with the antigens (100 µL/well in coating buffer) overnight
at 4 °C, covered with adhesive plate sealers. The next day, the plates
were washed four times with PBST (300 µL/well), and the solutions
of the analyte (50 µL/well in PBST; zero analyte is only PBST) and/or
the antisera (50 µL/well in PBST, 100 µL/well for the noncompetitive
assays) were added and incubated for 30 min at room temperature.
The plates were washed again as before, and a solution of antiIgG-
HRP (1/6000 in PBST) was added to the wells (100 µL/well) and
incubated for 30 min more at room temperature. The plates were washed
again, and the substrate solution was added (100 µL/well). Color
development was stopped after 30 min at room temperature with 4 N
H2SO4 (50 µL/well), and the absorbances were read at 450 nm.
Immunochemicals. Immunoreagents such as goat anti-rabbit IgG
coupled to horseradish peroxidase (antiIgG-HRP) and proteins such as
keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), con-
albumin (CONA), and ovalbumin (OVA) were obtained from Sigma
(St. Louis, MO). The conjugation of the haptens 2a-f and 4a-e to
BSA, CONA, and OVA by the MA method has already been reported
(46-48). The rest of the hapten-protein conjugates were prepared as
described below. All the protein conjugates were stored freeze-dried
at -40 °C, and unless otherwise indicated, working aliquots were stored
at 4 °C in 0.01 M PBS at 1 mg mL-1
.
Preparation of 2g-KLH (Immunogen) and 2g-BSA (Homologous
Antigen): ActiVe Ester (AE) Method. Following previously described
procedures (46), hapten 2g (4.63 mg, 10 µmol) was activated using
freshly prepared solutions of N-hydroxysuccinimide (NHS, 11.50 mg,
50 µmol) and dicyclohexylcarbodiimide (DCC, 41.26 mg, 100 µmol)
in anhydrous dimethylformamide (DMF, 120 µL) and reacted with the
proteins (BSA or KLH, 10 mg) in 0.2 M borate buffer (1.8 mL).
Coating Antigens 2g-BSA, 2g-CONA, and 2g-OVA: Mixed Anhy-
dride (MA) Method. According to previously described procedures (47,
49), freshly prepared solutions of tributylamine (14.1 µL, 19.8 µmol)
and isobutyl chloroformate (9.4 µL, 22.8 µmol) in anhydrous DMF
(40 µL) were used to activate hapten 2g (12.51 mg, 18 µmol) in the
same solvent (190 µL). After the solution was stirred for 45 min at
Combined CompetitiVe Immunoassay Studies. The ability of DEHA
to shift the equilibrium of the binding of the antiserum to the coating
antigen was analyzed by coating each of two consecutive columns of
the microtiter plates with the same coating antigen (1 µg mL-1).
Solutions of PBST (“zero DEHA”) and DEHA (5 µM) were added
(50 µL/well) to two consecutive columns. Serial dilutions of the cor-
responding antiserum were then added to both columns (1/500 to
1/32 000 in PBST, 50 µL/well). The mixture was incubated for 30 min
at room temperature, and the plates were processed as described before.
Coating antigen/antiserum combinations showing an inhibition of the
absorbance in the presence of DEHA higher than 10% for the same

Determination of Dealkylated Hydroxytriazines
J. Agric. Food Chem., Vol. 51, No. 1, 2003 159
antiserum dilution were chosen for further studies on competitive assays
(see below).
NoncompetitiVe Indirect ELISA. For each coating antigen/antiserum
combination selected, the avidity of the different antiserum versus the
coating antigens was determined by measuring the binding of serial
dilutions (1/500 to 1/32 000, 100 µL/well) of each antiserum to the
antigen-coated microtiter plates (10 µg mL-1 to 9 ng mL-1, 100 µL/
well). The plates were processed as described above. From these
experiments, concentrations for the coating antigens and the antisera
were chosen to produce around 0.7-1 unit of absorbance in 30 min.
Screening for CompetitiVe Indirect ELISAs. For each coating antigen/
antiserum combination selected, 12 serial dilutions of the analyte
(10 000 nM to 1 pM in PBST) were added (50 µL/well), followed by
the corresponding appropriately diluted antiserum (50 µL/well in PBST),
to the plates coated with the selected antigen concentration. The mixture
was incubated for 30 min, and the plates were then processed as
described above. The standard curve was fitted to a four-parameter
logistic equation.
To improve the immunoassay features, using the best coating antigen/
antiserum combination, a set of experimental parameters (detergent
concentration, ionic strength, length of the competition time, preincu-
bation effect, length of the coating step, and pH) were studied
sequentially as previously described (50).
Optimized CompetitiVe Indirect ELISA. Microtiter plates were coated
with 2g-CONA (1.25 µg mL-1 in coating buffer, 100 µL/well) and left
to stand for 24 h at 4 °C, covered with adhesive plate sealers. The
following day, the coated plates were washed with PBST (four times,
300 µL/well), and As72-DEHA solutions (As72 was diluted 1/500 and
DEHA at different concentrations from 5000 to 0.32 nM, both in PBST-
I) that had been preincubated for 1 h at room temperature were added
to the wells (100 µL/well) and incubated for 1 h at room temperature.
The plates were processed as previously described.
Figure 2. Synthetic pathway used to prepare hapten 2g.
while preserving the hydroxyl group. Moreover, previous studies
performed by our group showed that symmetric molecules were
highly recognized by antibodies raised against a hapten when
only one of the symmetric sides of the molecule was exposed
to the immunesystem (54). Therefore, when hapten 2g was used
as the immunizing hapten, there was also a chance for DEDIHA
to be recognized by the antibodies raised.
The high polarity of these kinds of substances was the main
problem arising during the investigation of several synthetic
approaches to prepare hapten 2g. As mentioned in the Introduc-
tion, the high solubility of these aminotriazines in aqueous media
and their low extraction efficiency with organic solvents has
been reported (55). Hence, in the first series of experiments, a
lack of reactivity was observed in most of the reactions assayed
due to the low solubility of these metabolites in the different
organic media. For the same reasons, when the reaction took
place, the desired product was obtained in low yield and its
isolation and purification from the reaction mixture was
extremely difficult. All these questions led us to the idea of
working with the amino group protected until the last step. This
strategy would surely reduce the polarity of the compound and
therefore would allow working in organic media using the
classical organic synthesis procedures. Therefore, we proposed
using cyanuric chloride (1) as starting material and sequentially
introducing the different groups by nucleophilic substitutions
of the chlorine atoms. The amino group would be introduced
in a protected form from the beginning, followed by the
introduction of the spacer arm. In the last steps, the third chlorine
atom would be substituted by a hydroxyl group, and finally the
protecting group would be removed (see Figure 2).
The trityl group, commonly used in peptide synthesis, was
selected as the protecting group since its hydrolysis takes place
only under acidic conditions (56, 57). Consequently, in the first
step, tritylamine (triphenylethylamine, 2) was introduced by
nucleophilic substitution of one chlorine atom of the cyanuric
chloride (1) to obtain compound 3. The reaction was performed
at -20 °C to avoid the substitution of more than one chlorine
atom. Subsequently, compound 5 was obtained after another
nucleophilic substitution reaction by 4-aminobutanoic acid 4
on a second chlorine atom of 1. Although such substitutions
usually take place at room temperature (46-48, 58), we needed
to heat the reaction mixture at 78 °C to add energy to the
chemical reaction, probably due to the steric hindrance resulting
from the great volume of the tritylamine group (58, 59). The
hydrolysis of the remaining chlorine atom has been reported to
occur upon heating the triazine herbicides in aqueous acidic
media (45). Consequently, the reaction conditions needed to
remove the protecting trityl group could also favor the replace-
ment of the chlorine atom by the hydroxyl group. As expected,
hapten 2g was obtained in 70% yield using a mixture TFA/
H2O/CH2Cl2 45:45:10. The overall yield of this synthetic
procedure from the cyanuric chloride 1 was 55% (see Figure
2).
Cross-ReactiVity Determinations. Stock solutions of the s-triazines
(desmetryne, prometrine, simazine, atrazine, DEA, DIA, and irgarol
1051) were prepared in DMSO (50 mM) and stored at 4 °C. Standard
curves for each of these compounds were constructed (5000 to 0.32
nM) in PBST-I. The preparation of the standard curves of the
dealkylated hydroxytriazines, 2-hydroxysimazine and 2-hydroxypro-
pazine, was done using the commercial stocks in acetonitrile (10 ng
µL-1). Each IC50 was determined in the competitive experiments
following the optimized protocol described above. The cross-reactivity
(CR) values were calculated according to the equation [IC50(DEHA)/
IC50(triazine)] × 100.
Accuracy Studies Using Blind Spiked Samples. This parameter was
assessed by preparing different blind spiked samples of DEHA in MilliQ
water. The samples were mixed with the antiserum, diluted in 0.1 M
PBS to obtain the appropriate ionic strength for the assay (0.05 M PBS,
70 mS cm-1). After the mixture was preincubated for 1 h at room
temperature, the samples were measured in the ELISA. Measurements
were performed in triplicate. The correlation was evaluated by
establishing a linear regression between the spiked and the measured
values.
RESULTS AND DISCUSSION
Hapten Synthesis. The design of the most suitable immuniz-
ing hapten has been considered the most crucial step in the
development of an immunochemical technique for a low-
molecular-weight analyte. The synthetic preparation of the
appropriate hapten can be sometimes tricky and time-consuming,
but the effort is worthwhile, considering that important features,
such as the specificity and selectivity of the resulting antibodies,
are mainly determined by the chemical structure of the im-
munizing hapten used to raise antibodies (36, 51-53). With
the idea of producing antibodies to recognize a broad range of
HADPs for screening purposes, we thought of hapten 2g
(compound 6) as a suitable chemical structure to produce
antibodies against DEHA, DIHA, and DEDIHA. The linker
substituting one of the amino groups would mimic the isopropyl
and ethyl groups present in DEHA and DIHA, respectively,

160 J. Agric. Food Chem., Vol. 51, No. 1, 2003
Sanvicens et al.
Table 2. Results from the Combined Competitive Experiments Used
Table 3. Immunoassay Features of the Competitive ELISAs Using
DEHA as Analyte
for Screening the Different Antiserum/Coating Antigen Combinationsa
r 2
a
coating
antigen
inhibitionb
(%)
absorbance
antiserum
72
coating antigen
Amax
Amin
slope
IC
50
antiserum
As71
no DEHA
5 µM DEHA
2f-BSA
0.89
1.14
0.40
1.37
0.47
1.34
0.31
0.54
0.21
0.20
0.11
0.02
−0.71
−0.77
−0.10
−0.98
−0.19
−0.63
29.01
5.52
4.31
17.03
5.03
20.10
0.97
0.97
0.97
0.90
0.96
0.91
2f-CONA
2g-BSA
2g-CONA
2g-BSA
2g-CONA
2f-BSA
2f-CONA
2d-CONA
2e-CONA
2f-BSA
0.419
0.733
0.631
0.481
0.731
1.067
1.693
0.555
0.386
0.425
0.503
0.719
1.244
1.592
0.618
1.952
0.341
1.09
0.418
0.702
0.618
0.408
0.548
0.701
1.643
0.541
0.119
0.122
0.413
0.682
1.009
1.446
0.561
1.703
0.138
0.378
0
4
2
15
25
34
3
As72
As73
73
2f-CONA
4a-BSA
a IC values are expressed in micrograms per liter. The parameters were
extracted from the four-parameter equation used to fit the standard curves.
50
2g-BSA (EA)
2g-BSA (MA)
2g-CONA (MA)
2e-BSA
2e-CONA
2f-BSA
2f-CONA
2d-OVA
2g-BSA (EA)
2g-BSA (MA)
2g-CONA (MA)
3
69
71
18
5
19
9
of nondesired side conjugation reactions had occurred when
using the active ester method, as reported (61). Those combina-
tions showing an inhibition of the assay titer higher than 10%
in the presence of the analyte were selected to perform two-
dimensional titration experiments to establish the appropriate
concentrations of the immunoreagents for the competitive
immunoassays. Table 3 shows the features of the indirect
ELISAs obtained. Immunoassay As72/2g-CONA was selected
for further optimization and evaluation because of its acceptable
detectability (limit of detection), slope, and the low background
noise.
9
13
60
65
a The experiments were performed by coating with each antigen two columns
of the plates at 1 µg mL-1 and adding serial dilutions (1/500 to 1/32000) of the
antiserum in the presence or in the absence of DEHA. b Inhibition was calculated
according to the following formula: [absorbance(no DEHA)/absorbance(5 µM
DEHA)] × 100.
ELISA Evaluation. Figure 3 shows the results of the studies
made to evaluate immunoassay performance. Concerning the
effect of the concentration of Tween 20, it was observed that,
while the maximal absorbance of the assay was not affected,
the detectability improved slightly with the concentration of the
detergent (see Figure 3A). Thus, a 0.1% concentration of Tween
20 was selected for further experiments. Regarding the ionic
strength effect, the detectability improved significantly from 0
to 30 mS cm-1 (20 mM in terms of PBS), remaining constant
between 30 and 70 mS cm-1 (50 mM in terms of PBS). The
maximal absorbance diminished from 0 to 15 mS cm-1 (10 mM
in terms of PBS), although it did not vary as much between 15
and 70 mS cm-1 (see Figure 3B). Therefore, 70 mS cm-1 was
the conductivity chosen to perform this assay. The length of
the competitiVe step was set at 60 min as a compromise, since
after this time the IC50 value increased, diminishing the
immunoassay detectability (see Figure 3C). Similarly, a pre-
incubation time of 60 min for the As72 and the analyte, before
their addition to the coated plate, was found to improve slightly
the immunoassay detectability (from 7.6 to 5.4 µg L-1). It was
also observed that a decrease of the length of the coating step
below 24 h led to a diminution of the immunoassay detectability.
Finally, studies on the effect of the pH showed a better tolerance
of the assay to basic than to acidic media. At pH values lower
than 7.5, the IC50 increased drastically (see Figure 3D).
A working protocol was established by taking into consid-
eration the results of these studies. The microplates were always
coated for 24 h at 4 °C. A preincubation step of the As72 with
DEHA for 60 min at room temperature was introduced before
the competitive step. The length of the competition was set at
60 min. The concentration of the PBS was increased from 10
to 50 mM in order to produce a conductivity value near 70 mS
cm-1. The pH was kept at 7.5, and the concentration of Tween
20 was increased to 0.1%. Figure 4 shows the standard curve
obtained under these conditions, and Table 4 summarizes the
parameters defining the calibration graph of the immunoassay
AS72/2g-CONA. The assay shows an IC50 value of 3.75 µg
L-1 and a limit of detection of 0.32 µg L-1 for DEHA.
Immunoassay specificity was evaluated by preparing standard
curves of different related triazines and measuring them in the
assay. As expected, only DIHA was better recognized in this
Development of an Indirect ELISA. Hapten 2g was
conjugated to KLH and to BSA following the active ester
method. The coupling reaction was verified by analyzing the
BSA derivative by MALDI-TOF-MS. We estimated a hapten
density of around 15 haptens covalently attached to each
molecule of BSA. Three rabbits were inoculated with 100 µg
of 2g-KLH and boosted each month for 6 months until no
significant increase in the antibody titer was observed. The
antiserum obtained from each rabbit was named As71, As72,
and As73, respectively.
To prepare coating antigens, this same hapten was conjugated
to BSA, CONA, and OVA using the mixed anhydride method,
to avoid potential interferences due to the potential side reactions
that may have occurred during the preparation of the immunogen
(60, 61). The competitors 2a-f and 4a-e (see Table 1 for
chemical structures) coupled to BSA, CONA, and OVA had
been previously prepared by this same method (46-48).
The avidity of the As71-73 versus the 36 competitors was
tested by using noncompetitive ELISAs (data not shown).
Antibody titers were especially high for the homologous 2g-
protein conjugates. The conjugates of the haptens 2a-f and 4a,b
were recognized on a lesser extent, and the conjugates of the
haptens 4c-e were not recognized at all, probably due to
important structural differences between the immunizing hap-
tens, such as the presence of bulky groups (methylthio and tert-
butylamino groups). The antiserum/coating antigens combina-
tions producing absorbance values higher than 0.5 were tested
on a combined competitive screening assay (see Experimental
Section) to determine the antiserum’s ability to recognize the
analyte at a constant concentration of 5 µM. All the experiments
were performed with DEHA as target analyte, since it has been
reported to be more frequently found in the environment than
DIHA (24). Only the binding of the As72 and As73 to certain
coating antigens, such as those from haptens 2f and 2g, was
significantly inhibited (see Table 2). It is worth noting that the
homologous competitor showed the highest inhibition, but only
when the coating antigen had been prepared by a different
coupling procedure, supporting the possibility that formation

Determination of Dealkylated Hydroxytriazines
J. Agric. Food Chem., Vol. 51, No. 1, 2003 161
Figure 3. Graphs showing the influence of several parameters on the performance of immunoassay As72/2g-CONA. Left axes indicate IC50 and Amax
(×10) of the assay. Right axes indicate Amax/IC (×100). (A) Effect of the detergent concentration. (B) Effect of the ionic strength. (C) Effect of the length
50
of the competitive step. (D) Effect of the pH. The data presented are extracted from the four-parameter equation used to fit the standard curve. Standard
curves were prepared using two well replicates.
Table 5. Cross-Reactivity Data Obtained To Characterize the
a
Specificity of the Immunoassay As72/2g-CONA for DEHA
IC
CR
(%)
50
s-triazine
DEHA
DIHA
DEDIHA
desmetryne
prometryne
irgarol 1051
atrazine
log P
pK
(nM)
22.16
7.27
1250
a
−0.97
−1.32
−2.18
1.06
1.41
1.24
1.03
0.69
0.24
6.56
6.70
6.75
3.82
3.91
4.53
2.35
3.10
7.68
7.59
7.50
2.44
2.55
100
305
4%
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
>5000
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
<0.4
simazine
Figure 4. Calibration curve of the immunoassay As72/2g-CONA for DEHA
obtained after optimizing the protocol. The data presented correspond to
the average and the standard deviation of six assays run on six different
days. The curves were run using well duplicates. See Table 4 for the
features of the optimized immunoassay.
2-hydroxypropazine
2-hydroxyatrazine
2-hydroxysimazine
DEA
−0.10
−0.45
0.17
DIA
−0.18
a Cross-reactivity (CR) is expressed as a percentage of the IC50 of the DEHA
a
Table 4. Features of the Optimized Immunoassay As72/2g-CONA To
Analyze DEHA
divided by the IC50 of the triazine. Log P and pK values have been introduced to
explain the lack of recognition observed for the different triazines evaluated.
a
Amin
0.02 ± 0.01
Amax
slope
1.09 ± 0.10
−0.87 ± 0.05
3.72 ± 0.22
0.32 ± 0.08
37.79 ± 3.67 to 0.79 ± 0.27
0.998 ± 0.002
recognized in the assay. The recognition pattern of the competi-
tors (see above) had already shown the great importance of the
simultaneous presence of a free primary amino group and the
hydroxyl group in the chemical structure of the analyte. Thus,
the absence of recognition of several hydroxytriazines, such as
hydroxyatrazine, hydroxypropazine, and hydroxysimazine, dem-
onstrated that the presence of a hydroxyl group alone was not
sufficient for antibody recognition. Looking at the pKa values
of these triazines, it can be observed that the pKa of the
hydroxylated triazines is around 7.5, and therefore both the
protonated and the anionic forms will coexist at equilibrium
during the competition step. However, the pKa value of DEHA,
DIHA, and DEDIHA are lower, which means that these
molecules will be in their anionic form at the assay pH.
Similarly, the presence of the free primary amino group alone
IC , µgL-1
50
LOD, µgL-1
dynamic range
r 2
a The parameters are extracted from the four-parameter equation used to fit
the standard curve. The data presented correspond to the average of six calibration
curves run on six different days. Each curve was built using two-well replicates.
assay than DEHA, since the ethyl group remaining in the
molecule is better mimicked by the spacer arm of the immuniz-
ing hapten. The results of the cross-reactivities obtained are
shown in Table 5. The assay is quite specific for the dealkylated
hydroxytriazines, since none of the other triazines assayed were

162 J. Agric. Food Chem., Vol. 51, No. 1, 2003
Sanvicens et al.
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Technol. 1997, 31, 1325-1333.
Table 6. Results from the Preliminary Accuracy Studies Performed
with the Immunoassay As72/2g-CONA
a
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spiked concn (nM)
measd concn (nM)
recovery (%)
380
200
150
25
370.37 ± 1.96
219.48 ± 3.55
142.58 ± 15.22
27.04 ± 3.42
97
110
95
108
a The accuracy was evaluated by preparing blind spiked samples in water and
analyzing them by the optimized immunoassay protocol. Each value corresponds
to the mean of three replicates.
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reactivity shown by DEA and DIA. Triazines such as
desmetryne, prometrine, and irgarol, with a methylthio group,
were not recognized at all. The same was observed for atrazine
and simazine. The low log P values of DEHA, DIHA, and
DEDIHA tested show their greater polarity compared to the
other triazines. Consequently, these analytes can probably
establish with the antibodies other kinds of interactions that may
predominate over those of more hydrophobic nature. Although,
as mentioned in the Introduction, it was expected that the
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immunizing hapten) would be highly recognized, this analyte
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Finally, to evaluate immunoassay accuracy of the ELISA,
blind spiked samples were measured. The results shown in
Table 6 indicate a good accuracy of the method to analyze water
samples, since all recovery values are close to 100%, proof that
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)
Conclusions. The analysis of highly polar molecules in water
samples at the trace level is often limited by the efficiency of
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Received for review April 30, 2002. Revised manuscript received
October 22, 2002. Accepted October 22, 2002. This work has been
supported by the EC Program (Contract QLRT-2000-01670) and by
CICYT (BIO2000-0351-P4-05 and AGL2001-5005-E).
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