Immunochemical determination of 2,4,6-trichloroanisole as the responsible agent for the musty odor in foods. 1. Molecular modeling studies for antibody production

Article abstract of DOI:10.1021/jf034003h

Nine antisera have been raised against 2,4,6-trichloroanisole (2,4,6-TCA) by immunizing them with three different haptens. With the spacer arm at the meta position, hapten A (3-(2,4,6-trichloro-3-methoxyphenyl)propanoic acid) preserved all of the functional groups of the target analyte. In hapten B (5-(2,4,6-trichlorophenoxy)pentanoic acid), the spacer was placed in the molecule substituting the methoxy group. Finally, hapten C (3-(3,5-dichloro-4-methoxyphenyl)propanoic acid) held the spacer arm at the para position instead of the chlorine atom of the target analyte. Using theoretical models, we have studied how the molecular geometry and the electronic distribution are affected by the introduction of the linker. The evaluation of the avidity of the resulting antibodies demonstrates that the orientation produced by the spacer arm must also be considered an essential aspect. The screening for competitive assays performed after synthesizing a battery of heterologous competitors has provided with these antibodies eight indirect enzyme-linked immunosorbent assays with acceptable properties. From the number of assays obtained, their maximal absorbance, their signal-to-noise ratio, the slope, and the IC₅₀ values obtained, it can be concluded that hapten C provided the best antibodies.

Full text of DOI:10.1021/jf034003h

3924 J. Agric. Food Chem. 2003, 51, 3924−3931
Immunochemical Determination of 2,4,6-Trichloroanisole as the
Responsible Agent for the Musty Odor in Foods. 1. Molecular
Modeling Studies for Antibody Production
NURIA SANVICENS, FRANCISCO SAÄ NCHEZ-BAEZA, AND M.-PILAR MARCO*
Department of Biological Organic Chemistry, IIQAB-CSIC, Jordi Girona, 18-26,
08034-Barcelona, Spain
Nine antisera have been raised against 2,4,6-trichloroanisole (2,4,6-TCA) by immunizing them with
three different haptens. With the spacer arm at the meta position, hapten A (3-(2,4,6-trichloro-3-
methoxyphenyl)propanoic acid) preserved all of the functional groups of the target analyte. In hapten
B (5-(2,4,6-trichlorophenoxy)pentanoic acid), the spacer was placed in the molecule substituting the
methoxy group. Finally, hapten C (3-(3,5-dichloro-4-methoxyphenyl)propanoic acid) held the spacer
arm at the para position instead of the chlorine atom of the target analyte. Using theoretical models,
we have studied how the molecular geometry and the electronic distribution are affected by the
introduction of the linker. The evaluation of the avidity of the resulting antibodies demonstrates that
the orientation produced by the spacer arm must also be considered an essential aspect. The
screening for competitive assays performed after synthesizing a battery of heterologous competitors
has provided with these antibodies eight indirect enzyme-linked immunosorbent assays with acceptable
properties. From the number of assays obtained, their maximal absorbance, their signal-to-noise
ratio, the slope, and the IC50 values obtained, it can be concluded that hapten C provided the best
antibodies.
KEYWORDS: Trichloroanisole; hapten design; molecular modeling; musty odor; wine; cork; immunoassay
INTRODUCTION
oak bark and in the stoppers throughout their production, storage,
and transportation (7). One of the most accepted hypotheses
points to their formation from chlorophenols, after microorgan-
ism detoxification processes occurring through methylation of
the phenolic group (8, 9). Chlorophenols have been used for
many decades as insecticides and wood and textile preservatives
but can also appear after the degradation of other biocides
frequently used in the past, such as lindane and hexachloroben-
zene (6), or as a consequence of bleaching processes using
chlorinating agents (6, 10). In fact, chlorophenols and chloro-
anisoles residues have been found not only in wine but also in
water, sediments, and several food products (11-15). Particu-
larly, pentachlorophenol (PCP), commercialized in Europe as
Raco, has been applied for many years on the cork oak base to
control insect plagues (16) and the formulation of this product
contained not only PCP but also 2,3,4,6-tetrachlorophenol and
2,4,6-triclorophenol (2,4,6-TCP). It has similarly been speculated
that chlorophenols also present in the cellar environment or in
the pallets where the wine bottles are stored could be absorbed
by the cork and metabolized to TCA by the corresponding
microorganisms (3, 17).
Cork has been traditionally used to prepare stoppers as
closures of champagne and wine bottles due to its peculiar
features (impermeability to air and liquids, ability to adhere to
glass surface, compressibility, resilience, and chemical inertness)
and its recognized positive influence in the wine and champagne
aromas. However, the use of cork stoppers has been associated
with an undesirable musty and moldy odor known as “cork
taint”. In the early 1980s, 2,4,6-trichloroanisole (2,4,6-TCA)
was reported to be the primary compound responsible for cork
taint in wine (1). Although other compounds, especially other
chloroanisoles, can contribute to the musty/earthy odor in wine
(2, 3), 2,4,6-TCA has been shown to have the lowest sensory
threshold, which varies from 4 to 10 ng L-1 in white wine and
50 ng L-1 in red wine (1, 2, 4, 5). Cork taint has become a
significant economic problem. It has been estimated that 2-5%
of the wine bottles are affected, leading to nearly $1 billion in
losses per year in the wine industry (6).
During the last two decades, many studies have been
performed in order to obtain a good understanding of the nature
and causes of the cork taint. The compounds associated with
this taint, including 2,4,6-TCA, are microbial metabolites
produced by naturally occurring microflora present on the cork
The analytical determination of 2,4,6-TCA in liquid media
and cork material has relied on chromatographic techniques such
as gas chromatography/ mass spectrometry (GC/MS) or GC with
electron capture detector (5, 7, 10, 18). However, cleanup/
preconcentration steps consisting of liquid-liquid extraction
* To whom correspondence should be addressed. Tel: +34 93 400 6171.
Fax: +34 93 204 5904. E-mail: mpmqob@iiqab.csic.es.
10.1021/jf034003h CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/03/2003

Immunochemical Determination of 2,4,6-Trichloroanisole
J. Agric. Food Chem., Vol. 51, No. 14, 2003 3925
4-chloro-methylphenoxyacetic acid; C4, 3-chlorophenoxyacetic acid;
C5, 3-(2-hydroxy-3,5,6-trichlorophenyl)-2-propenoic acid; C7, 3-(3-
hydroxy-2,4,6-trichlorophenyl)propanoic acid; C8, 3-(2-hydroxy-3,5,6-
trichlorophenyl)propanoic acid; C13, 3-(4-hydroxy-3,5-dichlorophenyl)-
propanoic acid; C14, 3-(2-hydroxy-3,6-dichlorophenyl)propanoic acid;
C15, 2,6-dichloro-3-hydroxyacetic acid; C16, 2,4-dichloro-5-hydroxy-
acetic acid; and C21, 3-hydroxy-2,4,6-trichlorophenylacetic acid has
already been reported (36, 37, 41). Hapten C6, 2-hydroxy-3,5,6-
trichlorobenzoic acid, as well as other chemical reagents were obtained
from Aldrich Chemical Co. (Milwaukee, WI). The synthesis of the
haptens A, B, and C is described below. The synthesis and the spectral
data of the haptens C9, C10, C12, C18-C20, and C22 are given as
Supporting Information. The chemical structures of the immunizing
and competitor haptens are shown in Figure 1.
with organic solvents followed by solid phase extraction
procedures are always required. Moreover, because of the
intrinsic volatility of the TCA and its ability to adsorb to the
solid surfaces, no agreement has been achieved regarding the
efficiency of the TCA extraction procedures (2). In the last years,
other approaches that may improve the reliability of the TCA
analysis have been evaluated such as solid phase microextraction
(19, 20) or supercritical fluid extraction (21). However, high-
throughput screening (HTS) methods based on economically
attainable and straightforward technologies are required for the
small and medium wine and cork producer companies. In this
context, immunochemical techniques could not only afford the
necessary detectability and specificity for the target analyte with
little sample pretreatment but also offer other advantages such
as their reliability, simplicity, and low cost (22-28). Moreover,
immunochemical techniques can easily be adapted to the
simultaneous analysis of many samples, constituting excellent
HTS methods.
Immunochemical methods are based on selective antibodies
binding to the target analyte. The production of antibodies to
low weight molecules, such as TCA, implies the preparation of
derivatives named haptens, which have to be coupled to larger
molecules in order to raise immunogenicity. The preparation
of optimum haptens as immunogens and competitors has been
regarded as the most crucial step in the development of an
immunochemical technique for small molecules. Many literature
examples prove that an appropriate hapten design determines
the features of the resulting antibodies, which mainly govern
the specificity and the selectivity of an immunochemical
technique (22, 29-33). Theoretical molecular models and
calculations can be useful tools to assist prediction of which
hapten will be the most appropriate to raise antibodies (34-
36). Similarly, they can be used to assess the influence of the
degree of heterology between the competitors and the analyte
(37, 38). With these precedents, in the present paper, we report
the studies made to rationalize the effect of the immunizing
hapten chemical structure on the features of the resulting
immunoassays against TCA.
3-(2,4,6-Trichloro-3-methoxyphenyl)propanoic Acid (3, Hapten A)
General Protocol. A mixture of CH3I (0.5 mL, 7.2 mmol) and dry
K2CO3 (0.975 g, 7.2 mmol) was added to a solution of the ester 1
(methyl 3-(3-hydroxy-2,4,6-trichlorophenyl)propanoate) (36) (0.50 g,
1.8 mmol) in anhydrous dimethylformamide (DMF) (7 mL) at room
temperature under Ar atmosphere. The mixture was stirred for 12 h at
room temperature, washed with H2O to eliminate the excess of CH3I,
and extracted with Et2O. The organic layer was then washed again with
water and aqueous 1 N HCl to eliminate the remaining DMF. Finally,
the resulting organic phase was dried with MgSO4, filtered, and
evaporated under reduced pressure. The crude product was purified by
column chromatography (silica gel, CH2Cl2:hexane 1:1) to obtain the
pure anisole 2 as a white solid (0.38 g, 72% yield). Methyl 3-(3-
methoxy-2,4,6-trichlorophenyl)propanoate (2): IR ν (KBr, cm-1): 1745
1
(CdO), 1458 (COO- st), 1176 (C-O st), 865 (ArC-H δ oop). H
NMR (200 MHz, CDCl3) δ (ppm): 2.56 (t, J ) 8 Hz, 2H, -CH2-
COO-), 3.24 (t, J ) 8 Hz, 2H, PhCH2-), 3.72 (s, 3H, -OCH3 ester),
3.88 (s, 3H, -OCH3 anisole), 7.36 (s, 1HAr meta). 13C NMR (75 MHz,
CDCl3) δ (ppm): 27.1 (-OCH3 ester), 31.7 (C-3), 51.7 (C-2), 60.5
(-OCH3 anisole), 127.4 (C-4′), 128.7 (C-2′), 129.8 (C-6′), 130.6 (C-
5′), 136.2 (C-1′), 151.4 (C-3′), 172.4 (C-1). EM m/z (%): 296 (M+,
10), 261 (M+-Cl+, 100), 219 (M+-C6H5+, 88), 159 (C10H7O2, 35), 123
(C6OCl, 78), 59 (C2H3O2, 76). Anal. calcd for C11H11Cl3O3: C, 44.40;
H, 3.72; Cl, 35.74. Found: C, 44.57; H, 3.93; Cl, 35.92. Following,
the methyl ester was hydrolyzed by dissolving 2 (0.10 g, 0.3 mmol) in
tetrahydrofuran (THF, 6 mL) in a round bottom flask provided with a
condenser and a magnetic stirrer. A solution of aqueous 1 N NaOH
(0.032 g, 0.8 mmol) was added, and the mixture was heated at 80 °C
for 3 h. The THF was evaporated, and the crude product was redissolved
with a NaHCO3 saturated solution (10 mL) and washed with Et2O.
The aqueous phase was then acidified and extracted with AcOEt, dried
with MgSO4, filtered, and evaporated under reduced pressure to obtain
hapten A as a white solid (88 mg, 93% yield). IR ν (KBr, cm-1): 2939
(COO-H st), 1714 (CdO), 1458 (COO- st), 1051 (C-O st), 865
EXPERIMENTAL SECTION
Chemistry. General Methods and Instruments. Thin-layer chroma-
tography (TLC) was performed on 0.25 mm, precoated silica gel 60
F254 aluminum sheets (Merck, Darmstadt, Germany). Unless otherwise
indicated, purification of the reaction mixtures was accomplished by
“flash” chromatography using silica gel as the stationary phase. 1H and
13C NMR spectra were obtained with a Varian Unity-300 (Varian Inc.,
Palo Alto, CA) spectrometer (300 MHz for 1H and 75 MHz for 13C) or
1
(ArC-H δ oop). H NMR (200 MHz, CDCl3) δ (ppm): 2.62 (t, J )
8 Hz, 2H, -CH2COO-), 3.26 (t, J ) 8 Hz, 2H, PhCH2-), 3.88 (s,
3H, -OCH3), 7.37 (s, 1HAr meta). Melting point, 102-105 °C.
1
on a Gemini 200 (199.975 MHz for H and 50.289 for 13C). Infrared
5-(2,4,6-Trichlorophenoxy)pentanoic Acid (6, Hapten B). A solution
of methyl 5-bromovalerate (0.59 g, 2.5 mmol) in dry acetone (10 mL)
was added dropwise to a mixture of 2,4,6-trichlorophenol (4) (0.50 g,
2.5 mmol) and dry K2CO3 (0.96 g, 3.4 mmol) in dry acetone (5 mL)
and placed in a round bottom flask provided with a magnetic stirring
bar, a Dimroth refrigerant, and a balanced addition funnel under Ar
atmosphere. The mixture was heated at 80 °C for 5 h until the
disappearance of the starting material by TLC. The solvent was then
evaporated, and the remaining crude product was suspended in 1 N
HCl (20 mL) and extracted with Et2O (2 × 30 mL). The organic layer
was then washed with a saturated solution of NaCl, dried with MgSO4,
filtered, and evaporated to obtain a crude oil that was purified by silica
gel flash chromatography using hexane:Et2O (4:1) as the mobile phase.
As a result, the methyl 5-(2,4,6-trichlorophenoxy)pentanoate (5) was
spectra were measured on a Bomen MB 120 FTIR spectrophotometer
(Hartmann & Braun, Que´bec, Canada). GC/MS was performed on a
MD-800 capillary gas chromatograph with an MS quadrupole detector
(Fison Instruments, VG, Manchester, U.K.), and the data are reported
as m/z (relative intensity). The ion-source temperature was set at 200
°C, and a 15 m × 0.25 mm i.d. × 0.15 mm (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, 80-220 °C (10 °C/min), 220 °C (10 min); injector
temperature, 250 °C.
Molecular Modeling and Theoretical Calculations. Molecular mod-
eling was performed using the Hyperchem 4.0 software package
(Hyperube Inc, Gainesville, FL). Theoretical geometries and electronic
distributions were evaluated for 2,4,6-TCA and potential haptens using
semiempirical quantum mechanics MNDO (39) and PM3 (40) models.
All of the calculations were performed using standard computational
chemistry criteria.
1
isolated pure as yellow oil (0.48 g, 61% yield). H NMR (200 MHz,
CDCl3) δ (ppm): 1.89 (m, 4H, -CH2-), 2.43 (t, J ) 6.8 Hz, 2H,
-CH2COO-), 3.69 (s, 3H, -OCH3), 4.00 (t, J ) 5.4 Hz, 2H, PhCH2-),
7.3 (2, 2HAr meta). 13C NMR (75 MHz, CDCl3) δ (ppm): 21.3 (C-3),
29.3 (C-4), 33.5 (C-2), 51.4 (-OCH3), 73.1 (C-5), 128.6 (C-2′, C-6′),
129.2 (C-4′), 130.0 (C-3′, C-5′), 150.4 (C-1′), 173.7 (C-1). Following,
Synthesis of the Haptens. The preparation of the haptens C1, 2,4,6-
thichlorophenoxyacetic acid; C2, 2,4,5-trichlorophenoxyacetic acid; C3,

3926 J. Agric. Food Chem., Vol. 51, No. 14, 2003
Sanvicens et al.
Figure 1. Chemical structures of the immunizing and competitor haptens used throughout this study.
the methyl ester was hydrolyzed as described above by treating the
ester 5 (0.1 g, 0.3 mmol) in THF (6 mL) with 1 N NaOH (0.032 g, 0.8
mmol) at 80 °C for 5 h. Finally, hapten B was obtained as a white
solid (67 mg, 75% yield). IR ν (KBr, cm-1): 2939 (COO-H st), 1714
Chemicals and Immunochemicals. Immunochemicals were obtained
from Sigma Chemical Co. (St. Louis, MO). The preparation of the
protein conjugates and the antisera is described below.
Buffers. Unless otherwise indicated, phosphate-buffered saline (PBS)
is 0.01 M phosphate buffer and 0.8% saline solution, and the pH is
7.5. PBST is PBS with 0.05% 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% TMB (tetra-
methylbenzidine) and 0.004% H2O2 in citrate buffer.
1
(CdO), 1458 (COO- st), 1051 (C-O st), 865 (ArC-H δ oop). H
NMR (200 MHz, CDCl3) δ (ppm): 1.93 (m, 4H, -CH2-), 2.49 (t,
J ) 6.8 Hz, 2H, -CH2COO-), 4.01 (t, J ) 5.4 Hz, 2H, PhCH2-), 7.3
(2, 2HAr meta). Melting point, 102-105 °C.
3-(3,5-Dichloro-4-methoxyphenyl)propanoic Acid (9, Hapten C).
From methyl-3-(3,5-dichloro-4-hydroxyphenyl)propanoate (7) (41),
methyl 3-(3,5-dichloro-4-methoxyphenyl)propanoate (8) was obtained
Preparation of the Immunogens and BoVine Serum Albumins (BSA)
Homologous Antigens. ActiVe Ester (AE) Method. Following described
procedures (42), haptens A, B, and C (60 µmol) were reacted with
freshly prepared solutions of N-hydroxysuccinimide (8.62 mg, 75 µmol)
and dicyclohexylcarbodiimide (30.90 mg, 150 µmol) in anhydrous DMF
(200 µL) for about 2 h at room temperature until the appearance of the
urea precipitate that was removed by centrifugation. The supernatant
of each solution was split in two and then added dropwise to a KLH
solution and to BSA solution (30 mg/each) in 0.2 M borate buffer (1.8
mL) and stirred for 3 h at room temperature. The protein conjugates
were purified by dialysis against 0.5 mM PBS (4 × 5 L) and milliQ
water (1 × 5 L) and stored freeze-dried at -40 °C. Unless otherwise
indicated, working aliquots were stored at 4 °C in 0.01 M PBS at 1
mg mL-1. Hapten densities of the BSA conjugates were determined
by MALDI-TOF-MS by comparing the molecular weight of the
standard BSA and that of the conjugates (results available as Supporting
Information).
Preparation of the Coating Antigens. Mixed Anhydride (MA) Method.
Following the described procedures (43, 44), the immunizing and
competitor haptens (15 µmol) were reacted with tributylamine (4 µL,
16.5 µmol) and isobutylchloroformate (3 µL, 18 µmol) in DMF (160
µL). The solution containing the activated hapten was then divided in
three equivalent fractions and added to the BSA, CONA, and OVA
(10 mg/each) solutions in 0.2 M borate buffer (1.8 mL). The conjugates
were purified as described above. As before, hapten densities of the
BSA conjugates were determined by MALDI-TOF-MS (results avail-
able as Supporting Information).
1
as a yellow oil (0.5 g, 72% yield). H NMR (300 MHz, CDCl3) δ
(ppm): 2.61 (t, J ) 7.2 Hz, 2H, -CH2COO-), 2.87 (t, J ) 7.2 Hz,
2H, PhCH2-), 3.68 (s, 3H, -OCH3 ester), 3.87 (s, 3H, -OCH3 anisole),
6.08 (s, 1H, -OH), 7.14 (s, 2HAr ortho). 13C NMR (75 MHz, CDCl3)
δ (ppm): 29.8 (-OCH3 ester), 35.1 (C-3), 51.8 (-OCH3 anisole), 128.7
(C-3′, C-5′), 129.1 (C-2′, C-6′), 138.1 (C-1′), 146.3 (C-4′), 172.7
(C-1). The hydrolysis of the ester yielded hapten C as a white solid
1
(0.30 g, 71% yield). H NMR (300 MHz, CDCl3) δ (ppm): 2.66 (t,
J ) 7.8 Hz, 2H, -CH2COO-), 2.87 (t, J ) 7.8 Hz, 2H, PhCH2-),
3.88 (s, 3H, -OCH3 anisole), 7.14 (s, 2HAr ortho). Melting point,
63-65 °C.
B. Immunochemistry. General Methods and Instruments. The
MALDI-TOF-MS (matrix-assisted laser desorption ionization time-of-
flight mass spectrometer) 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 Corporation
(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 (WTW, Weilheim, Germany). Polystyrene microtiter
plates were purchased from Nunc (Maxisorp, Roskilde, DK). Washing
steps were performed on a SLY96 PW microplate washer (SLT
Labinstruments GmbH, Salzburg, Austria). Absorbances were read on
a SpectramaxPlus (Molecular Devices, Sunnyvale, CA). The competi-
tive curves were analyzed with a four parameter logistic equation using
the software SoftmaxPro v2.6 (Molecular Devices) and GraphPad Prism
(GraphPad Sofware Inc., San Diego, CA). Unless otherwise indicated,
data presented correspond to the average of at least two well replicates.
Polyclonal Antisera. The immunization protocol was performed on
female New Zealand white rabbits weighing 1-2 kg, as previously
described (42). Rabbits 74, 75, and 76 were immunized with A-KLH,

Immunochemical Determination of 2,4,6-Trichloroanisole
J. Agric. Food Chem., Vol. 51, No. 14, 2003 3927
Figure 2. MNDO optimized geometries of the target analyte and haptens A−D in frontal (top) and side (bottom) views. Calculations have been made
using the corresponding amide derivatives of the haptens. Gray is carbon; green is chlorine; red is oxygen; magenta is hydrogen; and dark blue is
nitrogen.
rabbits 77-79 were immunized with B-KLH, and rabbits 88-90 were
immunized with C-KLH. The corresponding antisera (As) obtained were
named with the rabbit numbers. Evolution of the antibody titer was
assessed by measuring the binding of serial dilutions of the different
antisera to microtiter plates coated with A-BSA (AE) for As74-76,
with B-BSA (AE) for As77-79, and with C-BSA (AE) for As88-90.
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. Antisera were obtained by centrifugation
and stored at -40 °C in the presence of 0.02% NaN3.
Enzyme-Linked Immunosorbent Assay (ELISA) 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 day after,
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 (RT). 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 RT. The plates were washed
again, and the substrate solution was added (100 µL/well). Color
development was stopped after 30 min at RT with 4 N H2SO4 (50
µL/well), and the absorbances were read at 450 nm.
RESULTS AND DISCUSSION
Hapten Design. Four different chemical structures were
initially contemplated as potential immunizing haptens to raise
antibodies against TCA (see Figure 2, top). Hapten A with the
spacer arm at the meta position preserved the three chlorine
atoms and the methoxy group, but the introduction of the alkyl
in the aromatic ring could affect its electronic properties. In
hapten B, the methoxy group was replaced by the spacer arm
but the rest of the molecule remained unchanged. In contrast,
the chemical structures of the haptens C and D had substituted
one of the chlorine atoms by the spacer arms at either the para
or the ortho position, respectively. From these last two potential
haptens, hapten C maximized the exposure to the immune
system of the area defined by the methoxy group and the two
chlorine atoms in ortho, which appeared as one of the most
important epitopes.
Theoretical models and calculations have proven to be useful
tools to predict the suitability of a particular chemical structure
as immunizing hapten to raise antibodies against a nonimmu-
nogenic compound such as TCA (34, 36-38). Therefore, by
analyzing the features of these molecules at their minimum
energetic levels according to MNDO and PM3 models, it was
expected to obtain more objective data. One of the criteria
studied was the electronic distribution. According to it, small
differences could be observed in the punctual charges of the
aromatic rings as shown in Figure 3. Hapten A followed the
same pattern of electronic distribution as TCA although the total
charge was more negative, mainly because of the less positive
charges at the meta positions produced by the introduction of
the spacer arm in one of them. In contrast, if the spacer arm
was introduced instead of the methoxy group, like in hapten B,
the electronic resemblance to the analyte, regarding punctual
charges in the aromatic ring, was complete. From this point of
view, haptens C and D showed greater differences. The positions
lacking the chlorine atom were in these haptens much more
positive, and as a result, the overall charge was significantly
less negative for these haptens (see Figure 3, right bars). Thus,
considering electronic distribution, hapten B better mimicked
the behavior of the analyte. However, we also have to consider
Noncompetitive indirect ELISA was used for the screening of the
avidity of the 13 antisera obtained vs the 69 coating antigens by
measuring the binding of serial dilutions (1/1000 to 1/64 000) of each
antisera to the microtiter plates coated with different dilutions (10 µg
mL-1 to 9 ng mL-1) of each of the BSA, CONA, and OVA conjugates.
From these experiments, optimum concentrations for coating antigens
and antisera dilutions were chosen to produce around 0.7-1 units of
absorbance in 30 min.
CompetitiVe Indirect ELISAs. The avidity of 2,4,6-TCA to compete
with the different coating antigens for the antibody binding was
investigated by adding 12 serial dilutions of the analyte (10 000 nM to
1 pM, in PBST, 50 µL/well) to the coated plates followed by the
appropriately diluted As (50 µL/well). 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 equation according to
the following formula: Y ) [(A - B)/1 - (x/C)D] + B, where A is the
maximal absorbance, B is the minimum absorbance, C is the concentra-
tion producing 50% of the maximal absorbance, and D is the slope at
the inflection point of the sigmoid curve.

3928 J. Agric. Food Chem., Vol. 51, No. 14, 2003
Sanvicens et al.
Figure 3. Graph showing the punctual charges calculated at their minimum
energetic level using MNDO molecular models. Calculations have been
made using the corresponding amide derivatives to mimic the conjugated
haptens. R is H for TCA and haptens A, C, and D. Ris (CH ) CONH for
2 3
2
hapten B.
the effect that the introduction of the spacer arm could provoke
the spatial conformation of these molecules. Attending to this
aspect, while in the TCA molecule, the methoxy group appeared
placed in the same plane as the one defined by the aromatic
ring (see Figure 2, bottom); haptens A and B showed a different
geometry since the alkyloxy group had the tendency to move
away from this plane. In contrast, the geometry of the analyte
was better preserved in haptens C and D, which showed the
same behavior as the target analyte.
Figure 4. Synthetic pathways used for the preparation of the immunizing
haptens A−C. The synthesis of the starting materials 1 and 7 had already
been reported in our group by Galve and co-workers (36, 41).
These results made the selection of the most suitable
immunizing hapten difficult. Despite the greater electronic
similarity of hapten B, we were concerned about a possible drop
in the avidity of the antisera if this hapten was used, especially
considering the change in the geometry of the alkyloxy group
evidenced by the molecular models. The geometry of haptens
C and D was similar to that of TCA, but the absence of one of
the chlorine atoms was also worrying. From these two haptens,
hapten C was preferred since it orientated better to the immune
system, recognition of the area defined by the methoxy group
and the chlorine atoms in ortho. On the other hand, regardless
of the small changes in the electronic distribution, hapten A
was the only hapten preserving entirely the functional groups
of the target analyte. Consequently, we decided to assess the
results obtained from the theoretical models by raising antibodies
against the three haptens A, B, and C. The features of the
resulting immunoassays could help us to know which parameter
has more influence on the antibody qualities in addition to
providing a battery of antibodies with different properties.
Synthesis of the Haptens. Hapten A was prepared forming
the anisole of the methyl 3-(3-hydroxy-2,4,6-trichlorophenyl)-
propanoate (36) using methyl iodide in the presence of dry K2-
CO3. The hydrolysis of the resulting methyl ester gave hapten
A with a 67% global yield. Hapten B was obtained in only two
steps from 2,4,6-TCP by O-alkylation through a nucleophilic
substitution of a ω-halogenated alkanoic acid by the phenoxy
group generated in basic media. The hydrolysis of the ester 5
afforded the desired hapten on a 46% global yield. Finally,
hapten C was prepared as hapten A with a 51% yield by
substituting and first forming the methyl ether of methyl-3-(3,5-
dichloro-4-hydroxyphenyl)propanoate (41) and hydrolyzing the
methyl ester (see Figure 4).
the antibodies to the analyte, even if present only at the trace
level, by diminishing the affinity of the antibodies vs the
competitor (28, 33, 34, 46). Thus, taking advantage of the battery
of phenolic compounds previously synthesized in our group (36,
37, 41), a new set was prepared by selecting some of them and
converting them to the corresponding anisoles following the
same procedure applied for hapten A (see Figure 1 for chemical
structures).
Antibody Production. Haptens A, B, and C were conjugated
to KLH and BSA following the AE method. The coupling
reactions were verified by analyzing the BSA derivatives by
MALDI-TOF-MS and comparing the observed molecular weight
with that of the intact protein. An average of 21, 38, and seven
haptens covalently attached to each molecule of BSA was
estimated for the conjugates A-BSA (AE), B-BSA (AE), and
C-BSA (AE), respectively. Three rabbits were inoculated with
each immunogen using 100 µg of the corresponding hemocyanin
conjugate (A-KLH, B-KLH, and C-KLH) and boosted each
month for 6 months until no significant increase in the antibody
titer was observed. The antisera obtained from immunizing with
A-KLH were named As74, As75, and As76. The antisera As77,
As78, and As79 were the antisera obtained from the three rabbits
immunized with B-KLH. Finally, from immunizing with C-
KLH, the antisera identified as As88, As89, and As90 were
produced.
Screening of the Antiserum Avidity for the Immu-
noreagents. At the first instance, the avidity of the antisera vs
the battery of competitors (C9, C10, C12, C18-C20, and C22
coupled to BSA, CONA, and OVA by the MA conjugation
method) was tested on a simple experiment by measuring the
binding of serial dilutions of the antisera to plates coated with
the coating antigens at 1 µg L-1 (see Table 1). The antisera
against A-KLH showed a broad recognition of most of the
chemical structures tested as competitors. The highest avidity
was observed for those coating antigens with an ether group
(e.g., C1-C4, C9, C22, and haptens B and C) instead of a
Some competitive immunoassays work better under heter-
ologous conditions (45, 46). This means that the chemical
structure of the hapten used as the competitor is slightly different
from that of the immunizing hapten and therefore different to
that of the analyte. The theoretical idea is to favor binding of

Immunochemical Determination of 2,4,6-Trichloroanisole
J. Agric. Food Chem., Vol. 51, No. 14, 2003 3929
Table 1. Relative Aviditiesa of the Antisera Raised against TCA vs the Battery of Competitor Haptens
a The relative avidities have been expressed as the absorbance obtained when measuring the binding of the antisera 1/1000 times diluted in PBST to microplates coated
with the antigens at a concentration of 1 µg mL-1. See the legend in the right part of the table for the meaning of the shadow boxes.
hydroxyl group. Thus, haptens C6 or C5 were not recognized
at all, while C7 and C14 were only slightly recognized (see
chemical structures on Figure 1). In contrast to these results,
the antisera obtained against immunogens B-KLH and C-KLH
showed a very narrow avidity and the titers observed were also
lower. Thus, As77-79 raised against B-KLH only recognized
the competitors C1 and C9. The recognition of hapten C1 was
expected since it only differed from hapten B in the length of
the spacer arm. From the antisera against C-KLH, As88 showed
a different behavior from As89 and As90, whichgave very low
responses for most of the antigens. Nevertheless, As88 only
recognized those coating antigens containing haptens possessing
a high degree of homology with the immunizing haptens such
as haptens B, C, and C1 (see Table 1).
Table 2. Features of the Best Competitive Assays Obtained with the
Antisera Raised against the Three Immunizing Haptensa
IC
50
2
immunogen
As/antigen
Amax
Amin
slope (µgL-1
)
R
A-KLH
As76/C3-OVA
As76/C14-CONA 1.129 0.076 −0.51
0.950 0.038 −0.49
3.18
2.29
9.73
4.82
5.84
0.21
0.19
5.73
6.93
0.980
0.980
0.980
0.980
0.995
0.995
0.991
0.990
0.980
B-KLH
C-KLH
As78/C9-OVA
As88/C1-CONA
As88/C-BSA
As88/C-CONA
As88/C-OVA
As88/B-CONA
As88/B-OVA
1.600 0.328 −0.80
0.620 0.004 −0.61
1.054 0.029 −0.95
0.648 0.007 −0.91
0.618 0.017 −0.92
0.517 0.014 −1.25
0.500 0.010 −1.61
a Only those assays showing acceptable features were selected for further
Effect of the Immunizing Hapten on the Immunoassay
Properties. Those As/coating antigen combinations showing
acceptable titers were used to assess the ability of the analyte
to displace the equilibrium, after establishing the appropriate
concentration of each immunoreagent by two-dimensional
checkerboard titration experiments. Table 2 shows the features
of the indirect ELISAs obtained that exhibited adequate
characteristics (Amax 0.5-1.2 units of absorbance, slope > 0.5,
signal-to-noise ratio > 5) and IC50 values lower than 10 µg
L-1. The most noticeable result was that despite the low avidity
shown for the competitors, the antisera obtained against hapten
C gave the greater number of competitive immunoassays with
acceptable features. It must be noticed that these are the results
obtained from the screening using the standard protocol
described in the Experimental Section. It is possible to think
investigation (Amax 0.5−1.2 units of absorbance, slope > 0.5, signal-to-noise
ratio > 5) and IC50 values lower than 10 µg L-1
.
that by changing the experimental conditions and the physico-
chemical features (ionic strength, pH, incubation times, etc.)
other combinations could have rendered usable assays. This
could be especially probable for the antibodies raised against
hapten A, in light of the great number of competitors that were
recognized by these antibodies.
CONCLUSIONS
Electronic distribution and geometry are objective criteria to
assist immunizing hapten design although it is not always clear
which parameter will have a greater influence on the antibody

3930 J. Agric. Food Chem., Vol. 51, No. 14, 2003
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Received for review January 2, 2003. Revised manuscript received
May 6, 2003. Accepted May 8, 2003. This work has been supported by
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