Enzyme-Linked Immunosorbent Assays based on Rabbit Polyclonal and Rat Monoclonal Antibodies against Isoproturon

Article abstract of DOI:10.1021/jf035498d

This work describes the production and characterization of rabbit polyclonal antisera (pAb) and rat monoclonal antibodies (mAb) against isoproturon. Coating antigen and enzyme-tracer formats were developed. Standard curves for isoproturon were conducted either in 40 mM phosphate buffered saline (PBS) or in Milli-Q water. PAb 352 together with the best enzyme tracer revealed in the optimized ELISA (enzyme tracer format) a test midpoint of 1.06 ± 0.34 μg/L (n = 19, standard set up in Milli-Q water) with a detection limit of about 0.1 μg/L. The comparable ELISA with mAb IOC 7E1 had test midpoints of 0.07 ± 0.04 μg/L (n = 7, standards in Milli-Q water) and 0.11 ± 0.08 μg/L (n = 33; standards in 40 mM PBS). The limits of detection were about 0.003 and 0.01 μg/L in Milli-Q water and PBS, respectively. Noticeable cross reactivities (CRs) were seen with the major metabolites, namely 4-isopropylaniline, 4-isopropylphenylurea, and 1-(4-isopropylphenyl)-3-methylurea. With pAb 352, these CRs were 5%, 7%, and 31%, respectively, and with mAb IOC 7E1, they were 3%, 5%, and ca. 19%, respectively. All arylurea herbicides had only minor CRs, which ranged from no CR (e.g., chlorosulfuron) to a maximum of 3.3% (chlortoluron). Influences of organic solvents (methanol, ethanol, acetonitrile, and acetone) were evaluated. Both pAb- and mAb-based immunoassays showed the highest tolerance for methanol, up to 5%. Ethanol and acetonitrile could not be used above 2% without an influence on the assays. The same was true for acetone, although tested only in the mAb-based assay. Water samples of different origins and matrices were spiked and analyzed with these pAb and mAb ELISAs. The results demonstrated that these immunoassays are useful screening tools.

Full text of DOI:10.1021/jf035498d

2462 J. Agric. Food Chem. 2004, 52, 2462−2471
Enzyme-Linked Immunosorbent Assays Based on Rabbit
Polyclonal and Rat Monoclonal Antibodies Against Isoproturon
PETRA M. KRA¨ MER,*,†,‡ MARVIN H. GOODROW,§ AND ELISABETH KREMMER
Technische Universita¨t Mu¨nchen, Wissenschaftszentrum Weihenstephan, Lehrstuhl fu¨r O¨ kologische
Chemie und Umweltanalytik, Weihenstephaner Steig 23, D-85350 Freising, Germany, GSF-National
Research Center for Environment and Health, Institute of Ecological Chemistry, Ingolsta¨dter
Landstrasse 1, D-85764 Neuherberg (Oberschleissheim), Germany, and GSF-National Research Center
for Environment and Health, Institute of Molecular Immunology,
Marchioninistr. 25, D-81377 Munich, Germany
This work describes the production and characterization of rabbit polyclonal antisera (pAb) and rat
monoclonal antibodies (mAb) against isoproturon. Coating antigen and enzyme-tracer formats were
developed. Standard curves for isoproturon were conducted either in 40 mM phosphate buffered
saline (PBS) or in Milli-Q water. PAb 352 together with the best enzyme tracer revealed in the optimized
ELISA (enzyme tracer format) a test midpoint of 1.06 ( 0.34 µg/L (n ) 19, standard set up in Milli-Q
water) with a detection limit of about 0.1 µg/L. The comparable ELISA with mAb IOC 7E1 had test
midpoints of 0.07 ( 0.04 µg/L (n ) 7, standards in Milli-Q water) and 0.11 ( 0.08 µg/L (n ) 33;
standards in 40 mM PBS). The limits of detection were about 0.003 and 0.01 µg/L in Milli-Q water
and PBS, respectively. Noticeable cross reactivities (CRs) were seen with the major metabolites,
namely 4-isopropylaniline, 4-isopropylphenylurea, and 1-(4-isopropylphenyl)-3-methylurea. With pAb
352, these CRs were 5%, 7%, and 31%, respectively, and with mAb IOC 7E1, they were 3%, 5%,
and ca. 19%, respectively. All arylurea herbicides had only minor CRs, which ranged from no CR
(e.g., chlorosulfuron) to a maximum of 3.3% (chlortoluron). Influences of organic solvents (methanol,
ethanol, acetonitrile, and acetone) were evaluated. Both pAb- and mAb-based immunoassays showed
the highest tolerance for methanol, up to 5%. Ethanol and acetonitrile could not be used above 2%
without an influence on the assays. The same was true for acetone, although tested only in the
mAb-based assay. Water samples of different origins and matrices were spiked and analyzed with
these pAb and mAb ELISAs. The results demonstrated that these immunoassays are useful screening
tools.
KEYWORDS: Rabbit polyclonal antiserum; rat monoclonal antibodies; isoproturon; 4-isopropylanilin;
4-isopropylphenylurea; 1-(4-isopropylphenyl)-3-methylurea; urea herbicides; immunoassay; ELISA; water
INTRODUCTION
rye (2). The urea herbicides are slightly water soluble and can
easily migrate through the soil to crops and thus enter the food
chain. Depending on the particular rainfall conditions and soil
properties, the herbicides can also reach the groundwater. Due
to the absence of microbial activity, degradation processes are
very slow and thus, with accumulation, nontolerable levels may
be achieved in drinking water. The European Community has
set a MAC (Maximum Admissible Concentration) of 0.1 µg/L
for the individual pesticide and 0.5 µg/L for total pesticides
[EC 80/778/EEC (3) is now repealed and replaced by Council
Directive 98/83/EC (4)]. To monitor the urea herbicides in water,
solid-phase extraction techniques, followed by HPLC or GC/
MS, are commonly employed. Although chromatographic
methods are advantageous for multiresidue analysis, they need
preconcentration and/or derivatization steps before analysis. In
addition, they require the use of organic solvents, which are
environmentally hazardous. Thus, there is a desire to develop
Arylurea herbicides cause problems in the environment
because they are quite often found in surface water or rainwater
(1). Isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea, is the
most common representative of the urea herbicides for pre- and
post-emergence control of black grass, silky bent grass, wild
oats, annual meadow grass, ray grass, many broadleaf weeds
in spring and winter wheat, spring and winter barley, and winter
* Address correspondence to this author. GSF-National Research Center
for Environment and Health, Institute of Ecological Chemistry. Phone: 49-
89-3187-2772. Fax: 49-89-3187-3371. E-mail: kramer@gsf.de.
† Technische Universita¨t Mu¨nchen, Wissenschaftszentrum Weihen-
stephan, Lehrstuhl fu¨r O¨ kologische Chemie und Umweltanalytik.
‡ GSF-National Research Center for Environment and Health, Institute
of Ecological Chemistry.
§ Current address: 109 Almond Drive, Winters, California, 95694.
GSF-National Research Center for Environment and Health, Institute
of Molecular Immunology.
10.1021/jf035498d CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/03/2004

Polyclonal and Monoclonal Antibodies for Isoproturon
J. Agric. Food Chem., Vol. 52, No. 9, 2004 2463
Riedel de Hae¨n (Seelze, Germany; now available through Sigma-
Aldrich, Taufkirchen, Germany). Stock solutions were prepared in
ethanol with a concentration of 1 mg/mL and stored at 4 °C. 1-(4-
Isopropylphenyl)-3-methylurea, 4-isopropylphenyl)urea, and 4-isopro-
pylaniline were purchased from Dr. Ehrenstorfer, Augsburg, Germany.
Freund’s adjuvants (complete and incomplete), keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA), protein A, hydrogen
peroxide (H2O2; 30%), 3,3′,5,5′-tetramethylbenzidine (TMB), N,N-
dimethylformamide (anhydrous DMF), N,N′-dicyclohexylcarbodiimide
(DCC; 99%), N-hydroxysuccinimide (NHS; 97%), and goat anti-rabbit
(Fc specific) conjugated to HRP were from Sigma-Aldrich Chemie
GmbH, Schnelldorf, Germany. Horseradish peroxidase (HRP; EC
1.11.1.7, lyophilized, ca. 1000 U/mg) was purchased from Serva
Electrophoresis GmbH, Heidelberg, Germany.
Slide-A-Lyzer dialysis cassettes, needles and syringes, and goat anti-
mouse, Fc specific were purchased from Pierce, Rockford, IL. Mouse
anti-rat mAb TIB-172 (κ-specific) can be obtained from American Type
Culture Collection [(ATCC); Manassas, VA, USA]. Goat anti-rat
conjugated with HRP was purchased from Dianova, Hamburg, Ger-
many. Reagents for synthesis were obtained from either Aldrich
(Milwaukee, WI) or Sigma (St. Louis, MO).
Buffer salts (sodium hydrogen carbonate, sodium acetate, sodium
chloride, sodium phosphate, disodium hydrogen phosphate, dipotassium
hydrogen phosphate, potassium dihydrogen phosphate) and solvents
[methanol, ethanol, and dimethyl sulfoxide (dried DMSO, GR, max.
0.05% H2O)] were purchased from Merck (Darmstadt, Germany).
Ultrapure water was prepared by purifying demineralized water in
a Milli-Q (MQ) filtration system (Millipore, Eschborn, Germany) and
used for preparation of the standards and solutions and in some assays
for the determination of the zero concentration (100% control value).
Microtiter plates (96 well, MaxiSorp, NUNC, Wiesbaden, Germany)
were used in all assays.
Washing steps were carried out with a NUNC Immunowash
(Roskilde, Denmark), which was connected to a vacuum pump (KNF
Neuberger, Laboport, K&K Laborservice, Mu¨nchen, Germany). Ab-
sorbances were read with a ThermoMax microtiter plate reader
(Molecular Devices, Palo Alto, CA) at 450 nm (reference 650 nm).
The inhibition curves were analyzed with the four-parameter logistic
equation (Softmax Pro, Molecular Devices, Palo Alto, CA).
During the preparation of conjugates centrifugations were carried
out with a Heraeus Sepatech Biofuge 15 (Heraeus, Hanau, Germany).
Hapten Syntheses. Using the criteria previously developed by
Goodrow and Hammock (13), idealized immunogen haptens (I and II,
Table 1) and a potential coating/tracer hapten (III, Table 1) were
prepared from the appropriate aryl isocyanate (or isothiocyanate) and
amino acids. The two heterologous haptens, I and II, were used for
immunization and screening purposes. These isoproturon derivatives
mimic the structure of the target compound to which is covalently
attached an innocuous methylene handle that is terminated by a
carboxylic acid group to facilitate conjugation to proteins. Hapten III,
a thiourea mimic of the immunizing hapten except for a shorter
methylene protein-attachment handle, was prepared as an ideal coating/
enzyme tracer hapten. Very sensitive assays for isoproturon were thus
produced. Cross-reactivities of the numerous other arylurea herbicides
were negligible.
The ease of synthesis encouraged the evaluation of additional
structural modifications (IV-X) that would be most (or least) important
in target molecule recognition by the antibody (or antibodies). The
purity of each hapten was assessed by thin-layer chromatography (TLC)
employing 0.2 mm precoated silica gel 60 F254 on plastic sheets from
E. Merck (Darmstadt, Germany). Sample-spotted plates were eluted
with an ethyl acetate-hexane (1:1, v/v) plus 2% acetic acid solution,
viewed first under UV light (254 nm) and after iodine staining for
confirmation of purity. Identity and purity were further confirmed by
1H and 13C NMR [General Electric QE 300 spectrometer (Bruker NMR,
Billerica, MA) operating at 300.1 and 75.5 MHz, respectively] and
infrared (IR) spectra [Mattson Galaxy Series FTIR 3000 spectrometer
(Madison, WI)]. Mobile protons were checked with D2O.
rapid, inexpensive, sensitive, and environmentally safe screening
methods for analyses, that can be used as an “alarm” and can
rapidly detect the presence of pesticides in water. Enzyme-linked
immunosorbent assays (ELISAs) are known to be fast screening
methods and sensitive, quantitative tools for pesticide analyses
(e.g., urea herbicides). After the development of polyclonal
antibodies (pAb) for monuron, diuron, and linuron by Schneider
et al. (5) and monoclonal antibodies (mAb) for diuron by Karu
et al. (6), the aim of this study was to devise an additional assay
for isoproturon, hopefully selective for the target in the presence
of the other structurally related ureas. Several antibodies against
isoproturon have been developed. Lie´geois et al. (7) described
a quantitative ELISA method (coating antigen format) based
on mAbs, which allowed detection of isoproturon of 20-250
µg/L in aqueous solution, and in the range of 1 µg/g in soils.
Katmeh et al. (8) developed a direct competitive ELISA
procedure (based on sheep pAbs) for the determination of
isoproturon in water, with a detection limit of 0.03 µg/L. These
immunoreagents have also been used in enzyme immunoaffinity
chromatography as a rapid semiquantitative technique for the
screening of water samples (9). Ben Rejeb et al. (10) described
an indirect enzyme immunoassay (based on rabbit pAbs) for
the determination of isoproturon in water matrices (detection
limit of 0.02 µg/L) and used a method of antibody purification
to reduce the heterogeneity of the medium, when the test is
performed within complex surface water matrices. Mallat et al.
(11) demonstrated an optical immunosensor for isoproturon.
Sheep pAbs were used and a detection limit of 0.012 µg/L was
described. These antibodies though showed high CRs for diuron,
chlortoluron, and linuron (93%, 53%, and 46%, respectively).
Another assay for isoproturon was developed by GEC-Marconi
Materials Technology, Hirst Division, Herts, UK, which has
been evaluated for water and soil samples within a Joint
European Union research project (12). The Hirst assay uses an
antigen coating format that found cross reactivities of 4% and
<1%, respectively, for 1-(4-isopropylphenyl)-3-methylurea and
4-isopropylphenylurea, the two demethylated degradation prod-
ucts of isoproturon. The second test within this evaluation was
the commercially available test-kit EnviroGard Isoproturon Plate
Kit, Strategic Diagnostisc Inc., Newark, DE. The results of this
evaluation study showed that ELISA microtiter plates are of
interest for the determination of phenylureas in water samples
and soil extracts, notably because of the elimination of time-
consuming sample preparation. The results of both tests
significantly correlated with the HPLC values (12). Another
plate test-kit on the market is the Isoproturon Plate Kit,
Envirologix Inc., Portland, MA.
To obtain our own unlimited source of immunoreagents, we
have developed a new set of in-house reagents. All immu-
noreagents mentioned above are either not commercially avail-
able or, if they are, are formatted into ready-to-use test-kits.
These immunochemicals developed in-house will be used for a
new automated biosensor system for on-line and/or off-line on-
site field screening measurements.
To our knowledge, this new mAb is the first rat mAb used
successfully in the field of environmental analysis.
In addition, this is the only paper that includes all three major
metabolites of isoproturon, namely 4-isopropylaniline, 4-iso-
propylphenylurea, and 1-(4-isopropylphenyl)-3-methylurea, which
might also occur in environmental samples.
MATERIALS AND METHODS
Chemicals, Reagents, and Instruments. Standards of isoproturon,
diuron, monuron, chlorbromuron, linuron, neburon, fluometuron, fenu-
ron, metabromuron, metoxuron, and tebuthiruron were purchased from
1-(5-Carboxypentyl)-3-(4-isopropylphenyl)-1-methylurea (I). A
mixture of 0.355 g (2.00 mmol) of 4-isopropylphenylisocyanate, 0.362
g (2.00 mmol) of 6-methylaminohexanoic acid dihydrate, and 4.2 mL

2464 J. Agric. Food Chem., Vol. 52, No. 9, 2004
Table 1. Cross Reactivities of Haptens
Kra¨mer et al.
%CR
compd
isoproturon
I (immunogen hapten)
II
III (enzyme-tracer hapten)
IV
X
R
R
R
R
4
pAb (352)a
pAb (352)b
mAb (IOC 7E1)a
mAb (IOC 7E1)b
1
2
3
O
O
O
S
CH
CH
CH
CH
CH
H
H
H
H
H
CH(CH )
3 2
CH(CH )
CH(CH )
3 2
CH(CH )
100
345
199
9
100
396
184
13
100
89
41
0.05
1
100
69
25
0.08
0.7
3
3
(CH ) COOH
3
2 5
3 2
(CH ) COOH
3
2 3
(CH ) COOH
3
2 3
3 2
O
CH
CH COOH
CH(CH )
3 2
22
50
3
2
a Coating antigen format; coating Ag: I-BSA. b Coating antigen format; coating Ag: II-BSA.
(4.2 mmol) of 1.0 M NaOH was agitated for 2 h and filtered through
Celite, then the filtrate was acidified to pH 2. Crystallization of the
resultant solid from ethanol-water (1:2, v/v) provided 0.520 g (85%)
of I as white crystals: mp 95.5-96.5 °C; TLC Rf 0.32; IR (KBr) 3376
(s, NH), 1717 (vs, COOH), 1623 (vs, amide CdO), 1525 (vs, amide
II), 1244 (m, C-O), 814 (w, 1,4-disub) cm-1; 1H NMR (DMSO-d6) δ
12.0 (br, 1 H, OH), 8.09 (s, 1 H, NH), 7.35 (d, J ) 8.1 Hz, 2 H, ArH-
2,6), 7.08 (d, J ) 8.1 Hz, 2H, ArH-3,5), 3.27 (t, J ) 7.2 Hz, 2 H,
CH2-6), 2.90 (s, 3 H, NCH3), 2.80 (heptet, 1 H, CH), 2.20 (t, J ) 7.2
Hz, CH2-2), 1.5 (m, 4 H, CH2-3,5), 1.2 (m, 2 H, CH2-4), 1.17 (d, J )
6.8 Hz, 6 H, 2 CH3); 13C NMR (DMSO-d6) δ 174.5 (COOH), 155.6
(urea CdO), 141.8 (ArC-4), 138.5 (ArC-1), 125.9 (ArC-3,5), 120.3
(ArC-2,6), 48.0 (C-6), 34.4 (NCH3), 33.8 (C-2), 32.9 (CH), 27.3 (C-
5), 26.0 (C-4), 24.5 (C-3), 24.1 (2 CH3). COSY, HETCOR, and APT
836 (m, 1,4-disub) cm-1 13C NMR (DMSO-d6) δ 171.9 (COOH), 156.2
;
(urea CdO), 142.3 (ArC-4), 138.3 (ArC-1), 126.3 (ArC-3,5), 120.4
(ArC-2,6), 50.4 (C-2), 35.9 (NCH3), 33.1 (CH), 24.4 (2 CH3).
1-(3-Carboxypropyl)-1-methyl-3-phenylthiourea (V). Compound
V was prepared and worked up in the same manner as I but on a 10.0-
mmol scale from phenylisothiocyanate and 4-methylaminobutanoic acid
hydrochloride, providing 2.15 g (85%) of V as a white solid: mp 85.0-
86.0 °C dec with gas evolution; TLC Rf 0.36; IR (KBr) 3387 (m, NH
or OH), 3243 (m, NH or OH), 1716 (vs, COOH), 1527 (s, amide II),
1
1324 (s, CdS), 1242 (m, C-O) cm-1; H NMR (DMSO-d6) δ 12.20
(s, 1 H, OH), 8.98 (s, 1 H, NH), 7.3 (m, 4 H, ArH-2,3,5,6), 7.1 (m, 1
H, ArH-4), 3.78 (t, J ) 7.3 Hz, 2 H, CH2-4), 3.22 (s, 3 H, NCH3), 2.26
(t, J ) 7.3 Hz, 2 H, CH2-2), 1.84 (quin, J ) 7.3 Hz, 2 H, CH2-3) (with
added D2O the 12.20 and 8.98 ppm peaks were undetectable); 13C NMR
(DMSO-d6) δ 181.3 (CdS), 174.9 (COOH), 141.4 (ArC-1), 128.4 (ArC-
3,5), 126.6 (ArC-2,6), 125.1 (ArC-4), 52.5 (C-4), 39.1 (NCH3), 31.2
1
experiments confirmed H and 13C NMR assignments.
1-(3-Carboxypropyl)-3-(4-isopropylphenyl)-1-methylurea (II). This
urea was prepared on a 5.00-mmol scale by using 0.805 g of
4-isopropylphenylisocyanate and 0.806 g (5.25 mmol) of 4-methylami-
nobutanoic acid hydrochloride in 10.5 mL of 1.0 M NaOH according
to the procedure for I and provided 1.29 g (93%) of II as a white
powder: mp 134.5-135.5 °C dec with gas evolution; TLC Rf 0.23; IR
(KBr) 3343 (m, NH), 1715 (vs, COOH), 1657 (s, amide CdO), 1536
1
(C-2), 22.7 (C-3). HETCOR and APT experiments confirmed H and
13C- assignments.
1-(5-Carboxypentyl)-3-(4-chlorophenyl)-1-methylthiourea (VI)
and 1-(3-carboxypropyl)-3-(4-chlorophenyl)-1-methylurea (VII) were
synthesized as described by Schneider et al. (5).
1-(3-Carboxypropyl)-1-methyl-3-phenylurea (VIII). The reaction
of 4-(methylamino)butanoic acid dihydrate (1.15 g, 7.50 mmol), 16
mL of 1.0 M NaOH, and 0.85 mL (0.93 g, 7.8 mmol) of phenyliso-
cyanate under conditions and workup similar to I was followed by the
crystallization of the crude product from acetonitrile providing 1.47 g
(83%) of VIII as white crystals: mp 112.5-113.5 °C dec with gas
evolution; IR (KBr) 3371 (vs, N-H), 1694 (vs, COOH), 1624 (vs,
urea CdO), 1530 (s, amide II), 1242 (s, C-O) cm-1; 1H NMR (DMSO-
d6) δ 12.1 (br, 1 H, OH), 8.20 (s, 1 H, NH), 7.46 (d, J ) 8.2, Hz, 2 H,
ArH-2,6), 7.22 (dd, J ) 8.2 Hz, 7.2 Hz, 2 H, ArH-3,5), 6.92 (t, J )
7.2 Hz, 1 H, ArH-4), 3.30 (t, J ) 7.2 Hz, 2 H, CH2-4), 2.93 (s, 3 H,
NCH3), 2.23 (t, J ) 7.3 Hz, 2 H, CH2-2), 1.73 (t, J ) 7.2 Hz, 2 H,
CH2-3); 13C NMR (DMSO-d6) δ 174.7 (COOH), 155.6 (urea CdO),
140.9 (ArC-1), 128.4 (ArC-3,5), 121.9 (ArC-4), 120.1 (ArC-2,6), 47.6
(C-4), 34.5 (NCH3), 31.0 (C-2), 23.1 (C-3). COSY, HETCOR, and APT
(vs, amide II), 1231 (s, C-O), 833 (m, 1,4-disub) cm-1 1H NMR
;
(DMSO-d6) δ 12.2 (br, 1 H, OH), 8.13 (s, 1 H, NH), 7.35 (d, J ) 8.3
Hz, 2 H, ArH-2,6), 7.08 (d, J ) 8.3 Hz, 2 H, ArH-3,5), 3.29 (t, J )
7.2 Hz, 2 H, CH2-4), 2.91 (s, 3 H, NCH3), 2.81 (heptet, J ) 6.9 Hz, 1
H, CH), 2.22 (t, J ) 7.2 Hz, 2 H, CH2-2), 1.72 (quin, J ) 7.2 Hz, 2 H,
CH2-3), 1.17 (d, J ) 6.9 Hz, 6 H, 2 CH3); 13C NMR (DMSO-d6) δ
174.4 (COOH), 155.6 (urea CdO), 141.9 (ArC-4), 138.4 (ArC-1), 125.9
(ArC-3,5), 120.2 (ArC-2,6), 47.5 (C-4), 34.4 (NCH3), 32.9 (C-2), 30.9
(CH), 24.1 (2 CH3), 23.0. C-3). COSY, HETCOR, and APT experi-
1
ments confirmed H and 13C NMR assignments.
1-(3-Carboxypropyl)-3-(4-isopropylphenyl)-1-methylthiourea (III).
Employing the procedure for I, 4-isopropylphenylisothiocyanate, 0.355
g (2.00 mmol), 0.323 g (2.10 mmol) of 4-methylaminobutanoic acid
hydrochloride, and 4.2 mL of 1.0 M NaOH provided 0.570 g (97%) of
III as white flakes: mp 88.0-89.0 °C; TLC Rf 0.36; IR (KBr) 3408
(m, NH), 3207 (m, NH), 1711 (vs, COOH), 1532 (vs, amide II), 1326
(vs, CdS), 1214 (m, C-O), 830 (w, 1,4-disub) cm-1; 1H NMR (DMSO-
d6) δ 12.16 (s, 1 H, OH), 8.90 (s, 1 H, NH), 7.2 (m, 4 H, ArH-2,3,5,6),
3.77 (t, J ) 7.1 Hz, 2 H, CH2-4), 3.21 (s, 3 H, NCH3), 2.86 (heptet, J
) 6.8 Hz, 1 H, CH), 2.26 (t, J ) 7.1 Hz, 2 H, CH2-2), 1.83 (quin, J )
7.1 Hz, 2 H, CH2-3), 1.20 (d, J ) 6.8 Hz, 6 H, 2 CH3); 13C NMR
(DMSO-d6) δ 181.1 (CdS), 174.3 (COOH), 144.8 (ArC-4), 138.8 (ArC-
1), 126.2 (ArC-2,6 or -3,5), 125.7 (ArC-2,6 or -3,5), 52.1 (C-4), 38.7
(NCH3), 33.1 (CH), 30.9 (C-2), 24.1 (2 CH3), 22.4 (C-3). HETCOR
1
experiments confirmed H and 13C assignments.
1-(3-Carboxypropyl)-3-(3,4-dichlorophenyl)urea (IX). Employing
the procedure for I, a mixture of 1.36 g (6.5 mmol) of 90%
3,4-dichlorophenylisocyanate, 0.67 g (6.5 mmol) of 4-aminobutyric acid,
and 6.6 mL of 1.0 M NaOH gave crude IX. Recrystallization from
acetonitrile yielded 1.70 g (6.05 mmol, 93%) of IX as white needles:
mp 151.0-152.0 °C dec; IR (KBr) 3340 (s, NH), 3190 (s, NH), 1693
(s, COOH), 1642 (vs, urea CdO), 1577 (vs, amide II), 1222 (s, C-O)
cm-1; 1H NMR (DMSO-d6) δ 12.1 (s, 1 H, OH), 8.77 (s, 1 H, ArNH),
7.84 (d, J ) 2.4 Hz, 1 H, ArH-2), 7.44 (d, J ) 8.8 Hz, 1 H, ArH-5),
7.24 (dd, J ) 8.4 Hz, 2.4 Hz, 1 H, ArH-6), 6.35 (t, J ) 5.6 Hz, 1 H,
NH), 3.10 (dt, J ) 6.2 Hz, 6.6 Hz, 2 H, CH2-4), 2.24 (t, J ) 7.4 Hz,
2 H, CH2-2), 1.66 (quin, J ) 7.1 Hz, 2 H, CH2-3). A COSY experiment
1
and APT experiments confirmed H and 13C NMR assignments.
1-(Carboxymethyl)-3-(4-isopropylphenyl)-1-methylurea (IV). Emu-
lating I, a mixture of 0.178 g (2.00 mmol) of sarcosine, 2.2 mL of 1.0
M NaOH, and 0.322 g (2.00 mmol) of 4-isopropylphenylisocyanate
provided 0.416 g (83%) of IV as a white powder: mp 153.0-154.0
°C dec with gas evolution; TLC Rf 0.10; IR (KBr) 3401 (s, NH), 1721
(s, COOH), 1634 (s, amide CdO), 1536 (vs, amide II), 1214 (vs, C-O),
1
confirmed H assignments.
1-(5-Carboxypentyl)-3-(3,4-dichlorophenyl)-1-methylthiourea (X).
A mixture of 1.0 g (5.0 mmol) of 3,4-dichlorophenylisothiocyanate,
1.0 g (5.8 mmol) of 6-methylaminohexanoic acid dihydrate, and 1.0

Polyclonal and Monoclonal Antibodies for Isoproturon
J. Agric. Food Chem., Vol. 52, No. 9, 2004 2465
M NaOH to pH 10 was treated as per compound I. Crystallization of
the crude product from 1:1 (v/v) ethanol/water yielded 1.60 g (4.58
mmol, 92%) of X: mp 97.5-98.5 °C; TLC Rf 0.43; 1H NMR (DMSO-
d6) δ 12.0 (br, 1 H, OH), 9.10 (s, 1 H, NH), 7.63 (d, J ) 2.4 Hz, 1 H,
ArH-2), 7.54 (d, J ) 8.7 Hz, 1 H, ArH-5), 7.35 (dd, J ) 2.4, 8.7 Hz,
1 H, ArH-6), 3.77 (t, J ) 7.4 Hz, 2 H, CH2N), 3.22 (s, 3H, CH3N),
2.22 (t, J ) 7.3 Hz, 2 H, CH2-2), 1.6 (m, 4 H, CH2-3,5), 1.3 (m, 2 H,
CH2-4); 13C NMR (DMSO-d6) δ 180.5 (CdS), 174.6 (CdO), 141.5
(ArC-1), 130.1 (ArC-3), 129.7 (ArC-5), 127.1 (ArC-2), 126.2 (ArC-
4), 125.8 (ArC-6), 53.1 (C-6), 38.8 (N-CH3), 33.8 (C-2), 26.5 (C-5),
25.9 (C-4), 24.5 (C-3). A DEPT experiment revealed the 38.8 ppm
peak among the DMSO peaks.
Preparation of Hapten-Protein Conjugates for Immunogens and
Coating Antigens. Haptens I and II were conjugated to KLH and BSA,
respectively, according to Kra¨mer et al. (14). Briefly, the following
procedure was employed: 0.200 mmol of hapten I (MW 306.4) or
hapten II (MW 278.4), respectively, 0.202 mmol of NHS, and 0.223
mmol of DCC were dissolved in 1 mL of anhydrous DMF. The mixture
was stirred for 4 h at room temperature, followed by 12 h at 4 °C
(refrigerator). This mixture was centrifuged (6 min, 1400 rpm) to
remove the precipitate (urea). The clear supernatants were slowly (20
µL/10 min) pipetted to the stirred KLH and BSA solutions (50 mg/5
mL 0.05 M borate buffer, pH 7.8), respectively. Stirring was continued
for additional 16 h at 4 °C (refrigerator). The solutions were centrifuged,
and the clear supernatant was transferred to Slide-A-Lyzer cassettes
(Pierce, Rockford, IL) for dialysis against aqueous 0.2 M PBS (0.9%
(w/v) NaCl) for 4 days. Solutions were then freeze-dried and stored at
-25 °C. KLH conjugates were used for immunizations, and BSA
conjugates as coating antigens (pAb). For mAb development, BSA
conjugates were used for immunizations and KLH and BSA conjugates
utilized for screening.
Preparation of Hapten-Enzyme Conjugates for Enzyme Trac-
ers. Enzyme tracers were prepared according to the method described
by Schneider et al. (5). Briefly, 3 µmol of hapten I (MW 306.4), hapten
II (MW 278.4), or hapten III (MW 294.4) separately, 15 µmol of NHS,
and 30 µmol of DCC were dissolved in 130 µL of anhydrous DMF.
The solution was stirred for 4 h at room temperature followed by 12 h
of stirring at 4 °C (refrigerator). The solution was centrifuged (10 min,
1400 rpm), and the clear supernatant was added slowly to the enzyme
solution (2 mg of HRP in 3 mL of 130 mM sodium hydrogen carbonate,
pH 8.4). This solution was stirred overnight at 4 °C (refrigerator) and
centrifuged (10 min at 1400 rpm), and the clear solution was transferred
to Slide-A-Lyzer cassettes for dialysis against 130 mM sodium
hydrogen carbonate, pH 8.4, for 4 days. The solutions were then stored
in dark glass vials at 4 °C (refrigerator) and appeared to be stable for
at least 4 years.
Production of Polyclonal Antisera (pAb) for Isoproturon. Im-
munization of Rabbits and Screening of Sera. Six female Chinchilla
bastard rabbits (2.5 kg) were used for immunizations: three were
immunized with I-KLH conjugate and three with II-KLH conjugate.
Immunogens were dissolved in a sterile, 1.7% NaCl aqueous solution
at a concentration of 1 mg/mL. Conjugates (300 µL) and NaCl solution
(1200 µL) were mixed with 1500 µL of Freund’s complete (first
immunization) or Freund’s incomplete (all boost injections), respec-
tively, so that 100 µg/rabbit was injected. Boost injections were
performed at monthly intervals. All antisera were screened for anti-
isoproturon antibodies with the same haptens I and II conjugated to
BSA as coating antigens. For the last boost (14th), 300 µg/rabbit was
used. Ten days after the last boost, rabbits were bled and the clear sera
were stored either at -25 or -80 °C.
h, bound monoclonal antibodies were detected by using peroxidase-
labeled goat anti-rat IgG+IgM antibodies (Dianova, Hamburg, Ger-
many) and o-phenylenediamine as chromogen in the peroxidase
reaction. BSA was used as a negative control. Positive reacting
hybridomas were further tested for inhibition of HRP-labeled I, II, and
III derivatives, respectively. For this, microtiter plates were coated with
mouse anti-rat kappa (5 µg/mL) and mouse anti-rat lambda (5 µg/mL).
Tissue supernatants were added for 1 h. After washing with PBS, either
50 µL of an isoproturon solution (10 µg/L in PBS) or 50 µL of PBS
(as control) was added. After a 2-h incubation, HRP-labeled tracers
(50 µL, 1:100) were added without washing. After 30 min, the plates
were washed with PBS, and the substrate added. Eleven mAbs reacted
positively with the enzyme tracer and were inhibited by isoproturon in
binding the tracer.
The immunoglobulin type of monoclonal antibodies was determined
by using biotinylated anti-rat IgG subclass-specific monoclonal antibod-
ies (ATCC, Manassas, VA). The best clone, IOC 7E1 (rat IgG2a,κ),
was finally used for further studies because of its high affinity to
isoproturon.
ELISA-Coating Antigen Format with Polyclonal Antisera. Mi-
crotiter plates were coated with I-BSA or II-BSA, respectively (250
µL/well, 0.1 µg/mL in 0.05 M sodium carbonate buffer, pH 9.6,
overnight at 4 °C). The next day, the plates were washed with 4 mM
PBST (pH 7.4.-7.6), and 150 µL of isoproturon standard (different
concentrations in Milli-Q water) was added to each well. Then 50 µL
of pAb 352 (1:40 000 in 40 mM PBST (PBS buffer containing 0.05%
(v/v) Tween 20), pH 7.4-7.6) was added per well and the solutions
were incubated for 2 h at room temperature. The plates were washed
with 4 mM PBST, and 200 µL of goat anti-rabbit conjugated to HRP
(dilution 1:17 000 in 40 mM PBST, pH 7.4-7.6; Sigma-Aldrich,
Schnelldorf, FRG) was added to each well and incubated for 1 h at
room temperature. After another washing step (4 mM PBST), substrate
and chromogen for HRP were added (200 µL/well, H2O2/TMB in 0.1
M sodium acetate buffer, pH 5.5). The enzyme reaction was stopped
after 20-25 min with 50 µL of 2 M H2SO4/well. Microtiter plates were
read at 450 nm (reference 650 nm). Standard curves were calculated
with the 4-parameter equation.
ELISA-Coating Antigen Format with Monoclonal Antibody.
Microtiter plates were coated with I-BSA or II-BSA, respectively (250
µL/well, 0.1 µg/mL in 0.05 M sodium carbonate buffer, pH 9.6,
overnight, 4 °C). The next day, the plates were washed with 4 mM
PBST, pH 7.6, and 150 µL of isoproturon standard (different concentra-
tions in Milli-Q water)/well and 50 µL of mAb IOC 7E1/well (1:2000
in 40 mM PBST, pH 7.6) was added successively and incubated for 2
h at room temperature. After the washing step with 4 mM PBST, 200
µL of goat anti-rat-mAb-HRP/well (1:25 000 or 16 ng/mL in 40 mM
PBST, pH 7.6, Dianova, Hamburg, FRG) was added and incubated
for 1 h at room temperature. Another washing step was used to remove
unbound material, and 200 µL of substrate/chromogen (H2O2/TMB)
solution per well was added. The reaction was stopped after 10 min
with 50 µL of 2 M H2SO4/well and the microtiter plates were read at
450 nm (reference 650 nm).
Enzyme Tracer Format with Polyclonal Antisera. Microtiter plates
were coated with 250 µL/well of protein A (2 µg/mL in 0.05 M sodium
carbonate buffer, pH 9.6) and maintained overnight at 4 °C. The next
day, the plates were washed with 4 mM of PBST and 200 µL of anti-
isoproturon-pAb 352 (1:10 000 in 40 mM PBST, pH 7.6) was added
to each well and incubated for 2 h at room temperature. For the
inhibition step, plates were washed again, and 150 µL of standard
(prepared in Milli-Q water) or sample was added to the wells. After a
preincubation of 60 min, enzyme tracer III-HRP (50 µL, 1:1000 in 40
mM PBS, pH 7.6) was added to each well. Then in 30 min, the plates
were washed and 200 µL of HRP substrate/chromogen solution (H2O2/
TMB in 0.1 M sodium acetate buffer, pH 5.5) was added. The enzyme
reaction was stopped after 20-25 min with 50 µL of 2 M H2SO4 per
well and the absorbance was measured at 450 nm (reference 650 nm).
Standard curves were evaluated with the 4-parameter equation.
Enzyme-Tracer Format with Monoclonal Antibody. Microtiter
plates were coated with mouse anti-rat-antibodies (200 µL/well, mouse
anti-rat kappa TIB-172, 2 µg/mL in 0.05 M carbonate buffer, pH 9.6,
overnight, 4 °C, or 2 h at room temperature). The plates were washed
Production of Monoclonal Antibodies (mAbs) for Isoproturon.
Immunization of Rats, Primary Screening, Fusion, Secondary Screening.
Approximately 50 µg of I-BSA or II-BSA conjugates was injected
intraperitoneally (ip) and subcutaneously (sc) into LOU/C rats, using
CPG2006 (TIB MOLBIOL, Berlin, Germany) as an adjuvant. After a
2-month interval, a final boost was given ip and sc 3 days before fusion.
Fusion of the myeloma cell line P3X63-Ag8.653 with the rat immune
spleen cells was performed according to a standard procedure described
by Kremmer et al. (15). Hybridoma supernatants were tested in a solid-
phase immunoassay with I-KLH or II-KLH adsorbed on polystyrene
microtiter plates. Following incubation with culture supernatants for 1

2466 J. Agric. Food Chem., Vol. 52, No. 9, 2004
Kra¨mer et al.
with 4 mM PBST and rat mAb IOC 7E1 (150 µL/well; 1:2000 in 40
mM PBS, pH 7.6) was added. After an incubation for 2 h at room
temperature, plates were washed again (4 mM PBST), and then 100
µL/well of isoproturon standards was added (either set up in 40 mM
PBS or in Milli-Q water) and incubated for 1 h at room temperature.
Then 50 µL/well of enzyme tracer (III-HRP 1:500 or 1:1000 in 40
mM PBS, pH 7.6) was added and the mixtures were incubated with
shaking for another 30 min at room temperature. After the plates were
washed three times with 4 mM PBST, 150 µL/well of substrate/
chromogen solution for the HRP reaction (TMB/H2O2 in 0.1 M sodium
acetate buffer, pH 5.5) was added and incubated for 10 min at room
temperature. Finally, the reaction was stopped with 50 µL/well of 2 M
H2SO4 and the absorbance was read at 450 nm (reference 650 nm).
Standard curves were evaluated with the 4-parameter equation.
Enzyme Tracer Format in Different Organic Solvents. Tests for
solvent tolerances were performed with both the pAb and the mAb.
ELISA procedures were performed as described above, using the
protocols for the pAb and the mAb, respectively. The only difference
was that isoproturon standards were set up in different percentages (v/
v) of methanol, ethanol, acetonitrile, or acetone. Standard curves were
evaluated with the 4-parameter equation.
Analysis of Spiked Water Samples. Water samples from different
origins (tap water, creek water, river water, and pond water) were drawn.
The pH of the water samples ranged from 7.5 to 9.3. Water samples
were spiked in the following way: 30 µL of the corresponding standard
concentration was added to each water sample to a final volume of 3
mL; this means the spiked water samples contained 1% of the standard
solution. For the spike with 0.5 µg/L, the water sample contained only
0.5% of the standard solution (15 µL in 3 mL). Samples were run in
parallel with a standard curve for isoproturon on each microtiter plate.
Standards and samples were determined in quadruplicate. The assays
were performed as described previously in the paragraphs Enzyme
Tracer Format with Polyclonal Antisera and Enzyme Tracer Format
with Monoclonal Antibody. Samples measured with polyclonal anti-
serum were not identical with those measured with monoclonal
antibody. The concentrations of the spikes were chosen to fit into the
linear dose-response region of the standard curves, so that it was
possible to calculate the obtained results.
Table 2. Assay Optimization with Polyclonal Antisera Enzyme Tracer
Format
%CR
monuron
antiserum
dilution
enzyme tracer
dilution
IC50%
[µg/L]
diuron
347 1:10 000
352 1:20 000
352 1:10 000
352 1:10 000
I-HRP 1:1 000
I-HRP 1:2 000
II-HRP 1:8 000
III-HRP 1:1 000
0.70
0.95
0.96
0.57
0.5
0.5
0.3
0.5
0.9
0.7
0.3
0.3
herbicides except isoproturon (Table 4) have negligible cross-
reactivities in these assays.
Although different combinations of immunizing and enzyme
tracer hapten could be used, the best combination in optimized
assays were I-KLH for immunization and III-HRP as the
enzyme tracer. This was true for the pAb- and mAb-based
assays.
Coating Antigen Format. Screening of Antisera. For the
determination of the antisera titers, I-BSA and II-BSA were
used as coating antigens for screening the blood sera. Generally,
both haptens were recognized by all six antisera, but not all
sera showed inhibition with isoproturon. Nevertheless, several
sensitive assays for isoproturon were obtained with different
sera. With serum 347 (immunizing hapten II-KLH) the test
midpoint (IC50% value or the concentration of isoproturon that
inhibits 50% of the antigen-binding sites of the antibodies) of
the optimized standard curves for isoproturon was about 45 µg/L
when I-BSA was used as the coating antigen, and about 99 µg/L
when II-BSA was used (data not shown). With the sera 351
and 352 (immunizing hapten: I-KLH) the test midpoint of the
optimized assay was about 55 µg/L when serum 351 was used
and about 102 µg/L when serum 352 (using coating antigen
I-BSA) was used; the averages of assay midpoints for these
sera were about 78 µg/L (pAb 351) and 309 µg/L (pAb 352),
when coating antigen II-BSA was used. Although all of these
antisera were processed further, the most detailed characteriza-
tions focused on serum 352.
RESULTS AND DISCUSSION
Table 1 shows the results of the cross reactivities of haptens
I, II, III, and IV in the coating antigen format with serum 352.
These haptens were tested as cross reactants to aid in selecting
the best hapten for use as an enzyme tracer (Table 2). When
coating antigen I-BSA was used (Table 1, footnote a), the CRs
were 345% (I), 199% (II), 9% (III), and 22% (IV). When
coating antigen II-BSA was used (Table 1, footnote b), the CRs
were 396% (I), 184% (II), 13% (III), and 50% (IV).
Synthesis of Haptens. Haptens I and II were designed
specifically for immunizing purposes as they possess the ideal
features expressed earlier (13). They are exact representations
of the target structures with a chemically inert methylene handle
sufficiently long to allow flexibility of the target structure that
extends from the carrier protein. Note that the longer five-
methylene handle provided better test results (Table 1) than
the three-methylene handle, suggesting that handle length and/
or flexibility were important factors in assay utility. Hapten III,
a thiourea, was designed as a surrogate tracer hapten based on
the observation that the best coating/tracer hapten has previously
contained a shorter methylene handle, and thus less flexibility,
than does the immunizing hapten. The use of the thiourea moiety
was based on earlier success with the urea herbicides monuron
immunoassay (5), and diuron flow injection immunoaffinity
analysis (FIIAA) developments (16). The rationale for dimin-
ished recognition at the “active” site is the loss of H-bonding
by the thiourea with the active site as well as a steric rejection
due to the larger size of the sulfur moiety compared to oxygen.
Compounds with structure IV-X were available or synthe-
sized for the purpose of evaluating appendage recognition and
potential use for the assay. Thus, there was negligible recogni-
tion of all thiourea structures (III, V, VI, and X). A 4-isopro-
pylphenyl group was a required appendage for antibody
recognition (I-IV), whereas a phenyl or 4-chlorophenyl group
in this position of a urea or thiourea displayed negligible
recognition. It is not surprising then that the 13 other arylurea
Enzyme Tracer Format with Polyclonal Antisera. With
use of the information from the coating antigen format, enzyme
tracers were made with several haptens. Basically, haptens I,
II, and III were useful to different extents in the enzyme tracer
format. Although the CRs for the different haptens varied from
over 300% down to about 10%, the development of sensitive
assays was possible with antiserum 352, when all of these
derivatives were investigated as potential enzyme tracer haptens.
When all derivatives were compared and also the %CR for
monuron and diuron were determined, the assay with hapten
III as enzyme tracer hapten demonstrated a slightly better
inhibition curve for isoproturon (IC50% ) 0.6 µg/L; Table 2).
Hapten IV, which showed 22% CR (coating antigen I-BSA)
and 50% (coating antigen II-BSA) with antiserum 352 failed
to meet our criteria for an enzyme tracer hapten. A selection of
four assays is shown in Table 2, which could be used for the
determination of isoproturon at the lower ppb levels. In addition
to serum 352, serum 347 also displayed good sensitivity.
In summary, serum 352 (immunogen I-KLH) and enzyme
tracer III-HRP were selected for extensive evaluation, because

Polyclonal and Monoclonal Antibodies for Isoproturon
J. Agric. Food Chem., Vol. 52, No. 9, 2004 2467
this pair demonstrated the best combination of high absorbances,
IC50% value, and the lowest CR for monuron and diuron.
Table 3. Cross Reactivities of Haptens, Using Enzyme Tracer Format
with III-HRP
Screening of Monoclonal Antibodies. For screening of
positive clones of mAbs, I-BSA and II-BSA were evaluated as
coating antigens. Of the positive clones which recognized I-
and/or II-BSA, only one clone was characterized further,
because it showed very good inhibition with isoproturon [IOC
7E1 (sub class IgG2a,κ)]. The coating antigen format was
optimized with both coating antigens, I- and II-BSA. Both
coating antigens were recognized by the mAb IOC 7E1 and
could be used in assays for isoproturon. The IC50% for
isoproturon were very similar with both coating antigens, which
was ca. 1.2 µg/L. The CR pattern was similar to that of the
polyclonal sera, but here the amount of CR was generally lower
(Table 1, footnotes a and b). When using coating antigen I-BSA,
mAb IOC 7E1 had a CR of 89% for hapten I, 41% for II, 0.05%
for III, and 1% for IV. CR were even lower when coating
antigen II-BSA was used with the CR being 69% for I, 25%
%CR
pAb
mAb
compd
isoproturon
I (immunogen hapten)
X
R
R
R
R
4
(352)a (IOC 7E1)b
1
2
3
O CH CH
H
CH(CH ) 100
O CH (CH ) COOH H CH(CH ) 128
O CH (CH ) COOH H CH(CH ) 78
100
35
20
3
3
3 2
3 2 5 3 2
II
3
2 3
3 2
III (enzym tracer hapten) S CH (CH ) COOH H CH(CH )
6
0.4
2
3
2 3
3 2
IV
V
VI
VII
VIII
IX
X
O CH CH COOH
S
S
O CH (CH ) COOH H Cl
O CH (CH ) COOH H
O H (CH ) COOH Cl Cl
S
H
CH(CH ) 24
3
2
3 2
CH (CH ) COOH H
CH (CH ) COOH H Cl
H
<0.01
0.01
0.3
<0.01
0.02
0.2
3
2 5
3
2 5
3
2 3
H
<0.01
0.6
<0.01
0.6
3
2 3
2 3
CH (CH ) COOH Cl Cl
0.01
0.08
3
2 5
a Analyte in Milli-Q water for pAb. b Analyte in 40 mM PBS for mAb.
Table 4. Cross Reactivities of Metabolites and Pesticides, Using Enzyme Tracer Format with III-HRP
a Isoproturon set as 100%; b in 1% Methanol (Milli-Q water for pAb; 40 mM PBS for mAb); nd not determined

2468 J. Agric. Food Chem., Vol. 52, No. 9, 2004
Kra¨mer et al.
Figure 2. Representative standard curves with rat mAb IOC 7E1
(immunizgen I-BSA) and III-HRP as the enzyme tracer. Microtiter plates
were coated with TIB-172 (2 µg/mL in 50 mM sodium carbonate buffer,
pH 9.7); mAb IOC 7E1 (supernatant 1:2000 in 40 mM PBS, enzyme tracer
III-HRP 1:500 in 40 mM PBS). Standards were set up in Milli-Q water.
The standard curve was calculated according to the 4-parameter equation
y ) (A − D)/1 + (x/C)∧B)) + D with the following values: A ) 1.643; B
) 1.013; C ) 0.059 µg/L; D ) 0.017; n ) 4. The first concentration,
which is presented in the graph as 0.0001 µg/L, refers to 0 µg/L (Milli-Q
water).
Figure 1. Representative standard curve with rabbit pAb 352 (immunogen
I-KLH), using III-HRP as the enzyme tracer. Microtiter plates were coated
with Protein A (2 µg/mL in 50 mM sodium carbonate buffer, pH 9.7); pAb
352 1:10 000 in 40 mM PBST, enzyme tracer III-HRP 1:1000 in 40 mM
PBS. Standards were set up in Milli-Q water. The standard curve was
calculated according to the 4-parameter equation y ) (A − D)/1 + (x/
C)∧B)) + D with the following values: A ) 0.913; B ) 0.775; C ) 1.16
µg/L; D ) 0.021; n ) 2. The first concentration, which is presented in
the graph as 0.0001 µg/L, refers to 0 µg/L (Milli-Q water).
for II, 0.08 for III, and 0.7% for IV. It is very interesting to
point out that derivative III, which was later used successfully
as an enzyme tracer hapten, has a CR less than 1%. Again,
hapten IV, which has a slightly higher CR, was not useful as a
tracer hapten in the enzyme tracer format.
Standard Curve and Cross Reactivities. Polyclonal Anti-
serum. Figure 1 shows a representative standard curve for
isoproturon, using the serum 352 (dilution 1:10 000; produced
from immunizing conjugate I-KLH) and enzyme tracer III-HRP
(dilution 1:1000). The IC50% value was 1.06 ( 0.34 µg/L (n
) 19, standard set up in Milli-Q water). This is a slightly less
sensitive assay than a former development with sheep pAb
(Katmeh et al. (8)), and comparable to the assay in the indirect
format shown by Ben Rejeb et al. (10).
for the metabolites 4-isopropylaniline, 4-isopropylphenylurea,
and 1-(4-isopropylphenyl)-3-methylurea were lower, being 3%,
5%, and ca. 19%, respectively (Table 4).
Among all other urea herbicides tested, only chlortoluron
showed a slightly higher CR of about 3% (Table 4). Monuron
and diuron showed only minor cross reactivities (0.9% and
1.8%, respectively). Other relevant cross reactivities were not
seen (Table 4).
Although these mAbs were developed in rats, they can be
used in the same way as the usually used mouse mAbs. Only
the catching antibody or protein have to be adapted to these rat
antibodies. Here, we used either mouse anti-rat mAb TIB-172
(κ-specific) or protein G. For conventional ELISA on microtiter
plate, the use of culture supernatants was sufficient. For
examination of immunosensor formats, it is recommended that
protein G purified antibodies will be utilized.
Other candidates with related structures were assayed for
recognition, but only derivatives I-IV showed noticeable CRs
(Table 3).
Besides mAb IOC 7E1, a second clone, namely IOC 10G7
(sub class IgG2b,κ), showed a relatively good inhibition curve
with an IC50% of about 8 µg/L. Although this mAb was not
thoroughly characterized, it is both promising and selective for
use in immunoaffinity columns, as it showed only a 0.3% CR
for both monuron and diuron.
Effect of Organic Solvents on Isoproturon Immunoassay.
Figure 3 summarizes the effects of organic solvents, namely
methanol, ethanol, and acetonitrile, on the isoproturon immu-
noassay, using both pAb 352 and mAb IOC 7E1, respectively
(Figure 3A-C). Acetone was also tested with mAb IOC 7E1
(Figure 3D). Effects were always evaluated in comparison to
0% solvent on the same microtiter plates. The same enzyme
tracer III-HRP was used throughout all tests.
The isoproturon immunoassay with pAb 352 shows less
tolerance to organic solvents, especially when the changes in
IC50% values are considered. Methanol can be tolerated up to
5% without a significant change in IC50%, whereas as little as
2% ethanol or acetonitrile display an increase in the IC50%.
Organic solvents also show an effect on the maximum absor-
bance of the assays. Generally, the absorbance decreases with
the increase of organic solvent. It should be noted though, that
usually higher amounts of organic solvents can be used, when
the standards and the sample are set up in the identical amount
Cross reactivities for the major metabolites 4-isopropylaniline,
4-isopropylphenylurea, and 1-(4-isopropylphenyl)-3-methylurea
were relatively high, namely 5%, 7%, and 31%, respectively
(Table 4). All urea herbicides tested demonstrated only minor
cross reactivities. Most CRs observed were <1%, with average
values for monuron and diuron of 0.1% and 0.4%, respectively.
Only neburon showed a CR of ca. 1% (Table 4).
Standard Curve and Cross Reactivities. Monoclonal An-
tibody. Figure 2 shows a typical standard curve for isoproturon,
using the rat mAb IOC 7E1 (dilution of culture supernatant:
1:2000; here immunizing conjugate was I-BSA) and III-HRP
as enzyme tracer (dilution: 1:500). When the standards were
performed in Milli-Q water, the IC50% value was 0.07 ( 0.04
µg/L; n ) 7. When standards were set up in 40 mM PBS, the
IC50% value was 0.11 ( 0.08 µg/L (n ) 33). Both IC50%
values are about 10 times lower than the one obtained with
polyclonal serum.
Similarly as shown for pAb immunoassay, standard curves
for isoproturon could also be obtained with I- or II-HRP as
enzyme tracer, respectively (data not shown). These standard
curves were slightly less sensitive than with III-HRP, and also
higher amounts of antibody and enzyme tracer had to be used.
Therefore, III-HRP was utilized in all further optimizations.
In comparison to the polyclonal serum, the cross reactivities

Polyclonal and Monoclonal Antibodies for Isoproturon
J. Agric. Food Chem., Vol. 52, No. 9, 2004 2469
Table 5. Immunoassay Results of Water Samples from Several
Sources, Using Isoproturon PAb 352 (immunogen I-KLH) and Enzyme
Tracer III-HRP
amount
spiked
[µg/L]
amount
determined
[µg/L]a
water source
drinking water
(tap water)
pH
7.7
without spike
0.1
0.5
1.0
10.0
without spike
0.1
0.5
1.0
10.0
without spike
0.1
0.5
1.0
10.0
without spike
0.1
<0.08b
0.09 ± 0.09
0.51 ± 0.17
1.2 ± 0.5
10.2 ± 4.2
<0.03b
0.19 ± 0.04
0.59 ± 0.05
1.3 ± 0.3
5.5 ± 1.1
<0.08b
pond water
8.2
8.3
8.5
creek water,
English Garden, Munich
0.02 ± 0.02b
0.3 ± 0.22
0.63 ± 0.36
6.3 ± 0.29
<0.08b
0.14 ± 0.04
0.46 ± 0.03
0.86 ± 0.08
5.8 ± 2.0
River Isar, Munich
0.5
1.0
10.0
a n ) 4. b e limit of detection (of the corresponding standard curve on the
same plate as the samples).
the isoproturon immunoassay using mAb IOC 7E1. Without
spike, all water samples were analyzed as negative (below the
limit of detection). When isoproturon was analyzed with the
pAb immunoassay, the spiked concentrations in different water
matrices were generally found (recovery 93 ( 41%, see Table
5). Drinking water showed the best recovery data.
By using mAb IOC 7E1 the analyses of the water samples
could be performed at lower concentrations, which are especially
interesting for complying with the EU drinking water directive.
Water samples drawn from different locations and matrices were
spiked and analyzed. The analysis of tap water showed very
good correlations between the spiked and analyzed concentra-
tions (Table 6). With some other matrices, the recovery was
not as good. Thus it is recommended that the standard curves
be performed in the corresponding matrix. In the future, the
latter should be checked before screening by conventional
analysis for potential contaminations. Nevertheless, this immu-
noassay would be a very powerful tool for routine sensitive and
selective screening of certain water matrices.
General Remarks on the Availability of Immunoreagents.
For the success and the further development of the immunoana-
lytical field, the availability of antibodies and haptens (either
for the usage in the coating antigen or in the enzyme tracer) is
still a critical factor. Although immunoassay test-kits are
commercially available for nearly all relevant pesticides and
other environmental contaminants, the immunoreagents alone
are not. The latter are the ones needed for new and innovative
formats and assay developments. Even if some reagents are
available, agreements have to be signed that limit their usage
to research only. Up to a certain stage, this might be sufficient,
but to bring new immunochemical technologies into a wider
range of applications, for example, engineering companies have
to be involved. They typically do not have the biochemical
expertise in-house and need the access to immunoreagents to
guarantee the future customers the needed support. This is up
to now not the case. To get out of this “catch-22” situation, it
would be necessary to find a public storage place, through which
both antibodies and haptens would be available to everybody.
This might be accompanied by payments, so that the original
Figure 3. Effect of organic solvents on isoproturon immunoassay, using
pAb 352 and mAb IOC 7E1. Solvents: methanol (A), ethanol (B),
acetonitrile (C), and acetone (only mAb IOC 7E1 (D)). Dotted lines: pAb
352. Solid lines: mAb IOC 7E1. Key: b, IC50% pAb 352; 2, IC50%
mAb IOC 7E1; [, absorbance pAb 352; 9, absorbance mAb IOC 7E1.
Zero percent corresponds to Milli-Q-water when pAb 352 was tested, and
to 40 mM PBS when mAb IOC 7E1 was tested.
of solvent. This is especially the case when only the absorbance
is affected, but not the IC50%.
Analysis of Spiked Water Samples. Different sets of water
samples from different origins were spiked and analyzed either
with the isoproturon immunoassay using the pAb 352 or with

2470 J. Agric. Food Chem., Vol. 52, No. 9, 2004
Kra¨mer et al.
by Dzantiev et al. (21). In the latter, polyelectrolytes are used
for homogeneous or semihomogeneous immunoassay formats,
which can be carried out within 30 min (instead of several hours
for conventional ELISAs).
Table 6. Immunoassay Results of Water Samples from Several
Sources, Using Isoproturon MAb IOC 7E1 (immunogen I-BSA) and
Enzyme Tracer III-HRP
amount
spiked
[µg/L]
amount
determined
[µg/L]a
ACKNOWLEDGMENT
water source
drinking water
pH
7.5
without spike
0.01
0.05
0.1
0.5
1.0
5.0
without spike
0.01
0.05
0.1
0.5
1.0
without spike
0.01
0.05
0.1
0.5
1.0
5.0
without spike
0.01
0.05
0.1
0.5
1.0
without spike
0.01
0.05
0.1
0.5
1.0
without spike
0.01
0.05
0.1
0.5
1.0
<0.005b
We thank Edina Barsi, Martina Wiesner, Sylvia Oberleitner,
Matthias Englmann, Cristina-Mihaela Weber, and Annette
Franke for technical assistance. The personnel of the GSF animal
facilities is acknowledged. Dr. Fangshi Li [Fellow of the DAAD-
K.C. Wong fellowship of the German Academic Exchange
Services (DAAD)] contributed part of this work regarding cross
reactivities of the polyclonal sera. We also thank Shirley Gee
and Adam Harris for discussions regarding hapten conjugation,
and the Departments of Entomology and Chemistry for some
facilities (University of California, Davis, CA). This work was
presented in parts at the XIVth International Plant Protection
Congress, Jerusalem, Israel, July 25-30, 1999, the Euroanalysis
XI, September 3-9, 2000, the GDCh Chemiedozententagung,
Ko¨ln, Germany, March 10-13, 2002, and the International
Workshop on Biosensors for Food Safety and Environmental
Monitoring, Marrakech, Morocco, October 9-11, 2003.
(tap water)
0.01 ± 0.003
0.05 ± 0.01
0.1 ± 0.02
0.45 ± 0.06
0.8 ± 0.1
2.4 ± 0.3c
creek water 1
English Garden,
Munich
8.3
8.4
e0.005b
0.009 ± 0.007
0.03 ± 0.004
0.08 ± 0.02
0.62 ± 0.1
1.1 ± 0.6
creek water 2
Schwabinger Bach
English Garden,
Munich
<0.002b
0.003 ± 0.001
0.04 ± 0.02
0.06 ± 0.02
0.61 ± 0.49
1.1 ± 1.0
3.2 ± 1.4c
creek water 3
Nymphenburg Castle,
Munich
8.4
8.3
8.6
9.3
e0.003b
LITERATURE CITED
0.008 ± 0.005
0.08 ± 0.04
0.13 ± 0.03
0.98 ± 0.27
2.1 ± 0.5
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Received for review December 22, 2003. Revised manuscript received
February 25, 2004. Accepted February 26, 2004.
JF035498D