Biological monitoring of 2,4,5-trichlorophenol (I): preparation of antibodies and development of an immunoassay using theoretical models.

Article abstract of DOI:10.1021/tx025555h

Antibodies against 2,4,5-trichlorophenol have been prepared after theoretical and molecular modeling chemical studies of three potential immunizing haptens with the aim to find out the one mimicking best the target analyte. Competitive direct and indirect ELISAs have been developed after screening a battery of haptenized enzyme tracers and coating antigens, respectively. The relation between the degree of heterology of the competitor and the resulting immunoassay detectability has been investigated according to the electronic similarities of the competitor haptens with the target analyte taking in consideration their pK(a) values. These studies have been performed using theoretical and molecular modeling tools to find out their electronic distribution at their minimum energetic levels. The results suggest that the competitors should have a high homology to produced assays with good detectability values. On the other hand detectability improves when lowering the hapten density of the competitors. An indirect competitive ELISA has been finally selected for further investigation. The immunoassay has an IC(50) value of 0.6 microg L(-)(1) and a limit of detection of 0.084 microg L(-)(1). The selectivity of the assay is high in relation to other chlorophenols frequently present in real samples. In contrast, the brominated analogues may also be recognized with this assay.

Full text of DOI:10.1021/tx025555h

1360
Chem. Res. Toxicol. 2002, 15, 1360-1370
Articles
Biologica l Mon itor in g of 2,4,5-Tr ich lor op h en ol (I):
P r ep a r a tion of An tibod ies a n d Develop m en t of a n
Im m u n oa ssa y Usin g Th eor etica l Mod els
Mikaela Nichkova, Roger Galve, and M.-Pilar Marco*
Department of Biological Organic Chemistry, IIQAB-CSIC, J orge Girona,
18-26, 08034, Barcelona, Spain
Received May 8, 2002
Antibodies against 2,4,5-trichlorophenol have been prepared after theoretical and molecular
modeling chemical studies of three potential immunizing haptens with the aim to find out the
one mimicking best the target analyte. Competitive direct and indirect ELISAs have been
developed after screening a battery of haptenized enzyme tracers and coating antigens,
respectively. The relation between the degree of heterology of the competitor and the resulting
immunoassay detectability has been investigated according to the electronic similarities of
the competitor haptens with the target analyte taking in consideration their pKa values. These
studies have been performed using theoretical and molecular modeling tools to find out their
electronic distribution at their minimum energetic levels. The results suggest that the
competitors should have a high homology to produced assays with good detectability values.
On the other hand detectability improves when lowering the hapten density of the competitors.
An indirect competitive ELISA has been finally selected for further investigation. The
immunoassay has an IC50 value of 0.6 µg L-1 and a limit of detection of 0.084 µg L-1. The
selectivity of the assay is high in relation to other chlorophenols frequently present in real
samples. In contrast, the brominated analogues may also be recognized with this assay.
developed countries, their persistence has determined its
widespread distribution in the environment still today
and the repeated exposure of the general population to
low concentrations of these chemicals. Thus, chloro-
phenols have been identified as usual contaminants of
surface (4-7) and groundwater (8, 9) samples. Similarly,
trichlorophenols have been found to contaminate soils
and sediments (7, 10-12). Consequently, drinking water
may also contain 2,4,5-TCP between other chlorinated
phenolic compounds at low levels (7, 9, 13-15) and
significant levels accumulate in fish and seafood (5, 16).
Thus, a bioconcentration factor of 250-310 has been
estimated in fish and concentrations around 35-59 µg
L-1 have been detected in the tap water of certain
locations in Finland (15).
The general population is exposed through contami-
nated environment, textiles, leather goods, domestic
preservatives, and edible products (17, 18). Intake may
occur dermally, orally or via respiratory tract. Several
studies performed in Germany and in the U.S. show that
2,4,5-TCP can be found the in the urine of a high
percentage of the population (18-20). Thus, in 1994 the
frequency of detection of 2,4,5-TCP in the urine of adults
living in the U.S. was 20% with a 95th percentile and
maximum urinary concentrations of 3 and 25 µg L-1 (21).
Occupational exposure occurs through inhalation and
dermal contact with this compound at workplaces where
2,4,5-TCP is used or produced. The NIOSH (National
Institute of Occupational Safety and Health) statistically
In tr od u ction
Chlorophenol-related compounds have been used for
over 35 years as fungicides, pesticides, and for many
other agricultural, industrial, and commercial uses.
Particularly, 2,4,5-trichlorophenol (2,4,5-TCP) has been
an intermediate in the manufacture of industrial and
agricultural chemicals, such as the phenoxy acid herbi-
cides 2,4,5-T (2,4,5-trichlorophenoxyacetic acid), Silvex
[2-(2,4,5-trichlophenoxy)propionic acid, 2,4,5-TP], Ronnel
(o,o-dimethyl-o-2,4,5-trichlorophenyl phosphorothionate),
and sodium 2,4,5-trichlorophenate (1). Additionally, 2,4,5-
TCP and its salts have been used as antifungic and
antimicrobial preservatives for adhesive products, syn-
thetic textiles, rubber, wood, paints, in the leather
industry and in pulp and paper mills. All technical and
formulated products are contaminated in varying degrees
by the most toxic dioxin isomer, 2,3,7,8-tetrachlorodi-
benzo-p-dioxin (TCDD), at levels sometimes exceeding 1
ppm in chlorophenoxyacid herbicides (2) and 2,4,5-TCP
products (3). Chlorophenols are also formed during
combustion of organic material, in pulp and paper
industries using chlorine-bleaching processes, and as
byproducts during water chlorinating procedures.
Even though the discharge of chlorophenols to the
environment has decreased in the past decade for the
* To whom correspondence should be addressed. Phone: +34 93
4006171. Fax: +34 93 204 5904. E-mail: mpmqob@iiqab.csic.es.
10.1021/tx025555h CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/10/2002

Biological Monitoring of 2,4,5-Trichlorophenol (I)
Chem. Res. Toxicol., Vol. 15, No. 11, 2002 1361
estimated that 758 workers were potentially exposed to
2,4,5-TCP during a 3 years period NOES survey 1981-
1983 (22).
previous paper, we demonstrated the convenience of
using theoretical studies to choose the most appropriate
immunizing hapten to raise antibodies against 2,4,6-
trichlorophenol (44). Similarly, we have demonstrated
that the immunoassay specificity and detectability can
also be modulated by the chemical structure of the
competitor hapten (45, 46). With these precedents, we
report here the preparation of antibodies for 2,4,5-TCP
and the development of an immunoassay. Theoretical
models have been used with the aim to achieve sufficient
specificity and detectability to assess human exposure
in environmental and biological monitoring programs.
2,4,5-TCP is within the five chlorophenols out from 19
considered to have significant toxicological effects and
potential carcinogenicity (23). An oral Minimal Risk Level
(MRL) of 0.003 mg/kg/day would be applicable for an
intermediate exposure duration according to the updated
toxicological profile for chlorophenols of the Agency for
Toxic Substances and Disease Registry (ATSDR) of the
U.S. Department of Health and Human Services (24). A
recent study among U.S. men aged 30-60 demonstrated
that the occupational exposure to chlorophenol is a risk
factor for nasal and nasopharyngeal cancers closely
associated with the duration of exposure (25). On the
other hand, long-term use of chlorophenol polluted
household drinking water and consumption of contami-
nated fish have been correlated with certain symptoms,
related to those occurred after chlorophenol occupational
exposure, such as gastrointestinal and skin symptoms
(15).
2,4,5-TCP may also appear in urine of people exposed
to hexachlorobenzene (HCB) (26), hexachlorocyclohexane
(HCH, lindane) (27), certain organophosphorus insecti-
cides (21, 28), and chlorophenoxyacid herbicides (29).
Thus, detection of 2,4,5-TCP in urine may be an indicator
of exposure to chlorophenols and to the above-mentioned
substances frequently used in industry, commerce, ag-
riculture and in private households for a variety of
reasons (30). On the other hand, a continuous excretion
of 2,4,5-TCP may also indicate a risk for dioxin exposure
(3, 31, 32).
Determination of the actual chlorophenol intake by the
population would provide a reliable estimation of the
individual health risk due to environmental and oc-
cupational exposure. Laboratory methodsfor 2,4,5-TCP
determination are based on chromatographic techniques,
usually gas chromatography coupled to electron capture
(ECD) or mass spectrometry (MS). Thus, these method-
ologies have allowed detecting 2,4,5-TCP in human urine
and blood serum (33-35) at low ppb level, but lack of
the necessary speed for rapid screening the large number
of samples involved on continuous biomonitoring pro-
grams. Rapid, inexpensive, and reliable techniques are
essential for the routine risk assessment of human and
environmental exposure to hazardous chemicals. Immuno-
chemical methods may fulfill the efficiency requirements
due to their simplicity, specificity, high detectability, and
high throughput sample processing capabilities.
The key step of the development of an immunoassay
for small molecules is the hapten design (36-39). Both
chemical structures, of the immunizing hapten and of the
competitor, may play an important role on the immuno-
assay features. The introduction of the spacer arm in the
molecule may cause deformations of the molecular ge-
ometry as well as changes in its electronic distribution.
On the other hand, often the chemical synthesis of the
hapten is one of the most time-consuming steps of the
antibody production process. Therefore, the synthetic
effort necessary to produce antibodies against an organic
molecule should be carefully evaluated in relation to the
chances to obtain a good antibody. Nowadays, it is
possible to make computer-assisted theoretical and mo-
lecular modeling studies in order to predict the suitability
of a particular chemical structure as hapten to raise
antibodies against the target analyte (39-43). In a
Exp er im en ta l Section
Molecu la r Mod elin g a n d Th eor etica l Ca lcu la tion s. Mo-
lecular modeling was performed using the Hyperchem 4.0
software package (Hyperube Inc, Gainesville, FL). Theoretical
geometries and electronic distributions were evaluated for 2,4,5-
trichlorophenol and the haptens using semiempirical quantum
mechanics MNDO (47) and PM3 (48) models. All the calculations
were performed using standard computational chemistry cri-
teria. Theoretical calculations regarding pKa values were carried
out using the ACD/pKa 1.2 software package (Advanced Chem-
istry Development Inc., Toronto, ON, Canada) at the Depart-
ment of Analytical Chemistry (University of Lund, Sweden).
Syn th esis of th e Ha p ten s. The preparation of hapten A
[3-(2-hydroxy-3,5,6-trichlorophenyl)propanoic acid] and haptens
1 (2,4,6-trichlorophenoxyacetic acid), 2 (2,4,5-trichlorophenoxy-
acetic acid), 3 [3-(2-hydroxy-3,5,6-trichlorophenyl)-2-propenoic
acid], 5 [3-(3-hydroxy-2,4,6-trichlorophenyl)propanoic acid], 7
[3-(4-hydroxy-3,5-dichlorophenyl)propanoic acid], 8 [3-(2-hy-
droxy-3,6-dichloropheny)propanoic acid], 9 (3-hydroxy-2,6-
dichlorophenylacetic acid), and 10 (3-hydroxy-4,6-dichloro-
phenylacetic acid) has already been reported by our group (36,
45, 49). Hapten B (2,4,5-trichlorophenoxypentanoic acid) was
synthesized following the procedure described by Kramer et al.
(50). Hapten 4 (2-hydroxy- 3,5,6-trichlorobenzoic acid), as well
as other chemical reagents were obtained from Aldrich Chemical
Co. (Milwaukee, WI). Finally haptens 11 and 12 were prepared
as follows.
(1) Gen er a l Meth od s a n d In str u m en ts. Thin layer chro-
matography (TLC) was performed on 0.25 mm, precoated silica
gel 60 F254 aluminum sheets (Merck, Gibbstown, NJ ). Unless
otherwise indicated purification of the reaction mixtures was
accomplished by “flash” chromatography using silicagel as
1
stationary phase. H 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). Infrared spectra were
measured on a Bomen MB120 FTIR spectrophotometer (Hart-
mann & Braun, Que´bec, Canada).
(2) 3-Ch lor o-2-m eth ylp h en oxya cetic Acid 11. A solution
of methyl bromoacetate (0.35 mL, 3.7 mmol) was added dropwise
to a mixture of 3-chloro-2-methylphenol (500 mg, 3.51 mmol)
and anhydrous K2CO3 (600 mg, 4.35 mmol) in dry acetone (15
mL). The mixture was refluxed for 5 h until the initial product
was no more detected by TLC. The solution was acidified with
1 N HCl and extracted with Et2O. The organic phase was
washed with saturated NaCl, dried with MgSO4, filtered and
distilled under vacuum to obtain 680 mg of methyl 3-chloro-2-
1
methylphenoxyacetate (90% yield). H NMR (300 MHz, CDCl3)
δ 2.33 (s, 3H, -CH3), 3.79 (s, 3H, -OCH3), 4.59 (s, 2H, -CH2-),
6.66 (dd, J ) 8.8 Hz, J ) 3 Hz, 1HAr ortho), 6.79 (d, J ) 3 Hz,
1HAr ortho), 7.22 (d, J ) 8.7 Hz, 1HAr meta). 13C NMR (75 MHz,
CDCl3) δ 20.2 (-CH3), 52.1 (-OCH3), 65.4 (C-2), 113.1 (C-2′), 117.3
(C-6′), 126.9 (C-4′), 129.6 (C-3′), 137.2 (C-5′), 156.3 (C-1′), 169.1
(C-1). A solution of 0.5 N NaOH (17 mL, 8.5 mmol) was added
to a solution of the ester (300 mg, 1.39 mmol) in THF (20 mL).
The reaction mixture was stirred for 5 h at room temperature
until the complete disappearance of the starting material by
TLC. The solvent was evaporated and the residue dissolved in

1362 Chem. Res. Toxicol., Vol. 15, No. 11, 2002
Nichkova et al.
0.5 N NaOH (aq), washed with Et2O and acidified with 1 N HCl.
The aqueous layer was extracted with ethyl acetate, dried with
MgSO4, filtered, and evaporated to dryness under reduced
pressure to obtain 270 mg of the desired acid 11 (96% yield).
1H NMR (300 MHz, DMSO-d6) δ 2.28 (s, 3H, -CH3), 4.64 (s, 2H,
-CH2-), 6.76 (dd, J ) 9 Hz, J ) 3 Hz, 1HAr ortho), 6.92 (d, J )
3 Hz, 1HAr ortho), 7.27 (d, J ) 9 Hz, 1HAr meta). 13C NMR (75
MHz, DMSO-d6) δ 19.5 (-CH3), 64.6 (C-2), 113.5 (C-2′), 117.2
(C-6′), 126.9 (C-4′), 129.3 (C-3′), 136.3 (C-5′), 156.4 (C-1′), 169.7
(C-1).
4-hydroxycinnamic acid, 10 mg mL-1 in CH3CN/H2O 70:30, 0.1%
TFA) with 2 µL of a solution of the conjugates or proteins (10
mg mL-1 in CH3CN/H2O 70:30, 0.1% TFA).
P olyclon a l An tiser a . Three female New Zealand white
rabbits (As53-As55) were immunized with A-KLH and other
three rabbits (As56-As58) were immunized with B-KLH as
reported (43). Evolution of the antibody titer was assessed by
measuring the binding of serial dilutions of the antisera to
microtiter plates coated with A-BSA and B-BSA. After an
acceptable antibody titer was observed, the animals were
exsanguinated and the blood collected on vacutainer tubes with
a serum separation gel. Antiserum was obtained by centrifuga-
tion and stored at -80 °C in the presence of 0.02% NaN3. The
antisera (As) were used without further purification. Working
aliquots were stored at 4 °C.
Im m u n och em istr y. (1) Gen er a l Meth od s a n d In str u -
m en ts. The pH and the conductivity of all buffers and solutions
were measured with a pH-meter pH 540 GLP and a conduc-
timeter LF 340, respectively (WTW, Weilheim, Germany). The
centrifuge, model 5415D was from Eppendorf (Germany). Poly-
styrene microtiter plates were purchased from Nunc (Maxisorp,
Roskilde, D.K.). Washing steps were carried out using a SLY96
PW microplate washer (SLT Labinstruments GmbH, Salzburg,
Austria). Absorbances were read on a SpectamaxPlus microplate
reader (Molecular Devices, Sunnyvale, CA). The competitive
curves were analyzed with a four-parameter logistic equation
using the software SoftmaxPro v2.6 (Molecular Devices) and
GraphPad Prism (GraphPad Software Inc., San Diego, CA).
Unless otherwise indicated, data presented correspond to the
average of at least two well replicates.
(3) 2-Ch lor op h en oxya cetic Acid 12. As described for the
compound 11, a solution of methyl bromoacetate (0.38 mL, 3.9
mmol) was added to a mixture of 2-chlorophenol (500 mg, 3.89
mmol) and anhydrous K2CO3 (650 mg, 4.71 mmol) in dry acetone
(16 mL) to obtain 740 mg of methyl 2-chlorophenoxyacetate (95%
yield). 1H NMR (300 MHz, CDCl3) δ 3.80 (s, 3H, -OCH3), 4.61
(s, 2H, -CH2-), 6.79 (d, J ) 8.2 Hz, 1HAr ortho), 6.90 (s, 1HAr
ortho), 6.97 (d, J ) 8 Hz, 1HAr para), 7.20 (dd, J ) 8 Hz, J ) 8
Hz, 1HAr meta). 13C NMR (75 MHz, CDCl3) δ 52.2 (-OCH3), 65.3
(C-2), 113.0 (C-2′), 115.2 (C-6′), 122.0 (C-4′), 130.3 (C-3′), 135.0
(C-5′), 158.4 (C-1′), 168.9 (C-1). As described before, a solution
of 0.5 N NaOH (18 mL, 9 mmol) was added to a solution of the
ester (300 mg, 1.5 mmol) in THF (22 mL), and 250 mg of the
1
desired acid 12 were obtained (90% yield). H NMR (300 MHz,
DMSO-d6) δ 4.70 (s, 2H, -CH2-), 6.89 (d, J ) 8.5 Hz, 1HAr ortho),
6.98 (s, 1HAr ortho), 6.99 (d, J ) 8.5 Hz, 1HAr para), 7.29 (dd, J
) 8.5 Hz, J ) 8.5 Hz, 1HAr meta). 13C NMR (75 MHz, DMSO-
d6) δ 64.7 (C-2), 113.5 (C-2′), 114.6 (C-6′), 120.9 (C-4′), 130.6
(C-3′), 133.6 (C-5′),, 158.6 (C-1′), 169.6 (C-1).
P r ep a r a tion of P r otein Con ju ga tes. Immunogens: Hap-
tens A and B were covalently attached through their carbox-
ylic group to the lysine residues of KLH (keyhole limpet
hemocyanin) by the mixed anhydride (MA) method as described
by Ballesteros et al. (43). Briefly, each hapten (hapten A, 15.5
mg, 0.04 mmol and hapten B 15.5 mg, 0.04 mmol) was reacted
with tributylamine (10.5 µL, 0.04 mmol) and isobutylchloro-
formate (6.4 µL, 0.048 mmol) in anhydrous DMF (dimethyl-
formamide, 200 µL) and added to KLH (10 mg). Simultaneously,
BSA (bovine serum albumin) conjugates were prepared by the
same procedure reacting hapten A (20 µmol) and hapten B (20
µmol) with 10 mg of the protein. Enzyme tracers (ET): Haptens
1-5 and 7-12 (10 µmol) were coupled to HRP (2 mg) using the
active ester (AE) method as reported (43, 44). Coating antigens
(CA): Active Ester (AE) Method. Following described procedures
(45, 49, 51) the haptens 1-3, 5, and 7-12 (10 µmol) were
covalently attached to BSA, OVA (chicken egg ovalbumin) and
CONA (chicken egg conalbumin) (10 mg/each) in a hapten:
protein (lys) molar ratio 2:1. Haptens 5 and 7 were conjugated
to BSA and OVA using different hapten:protein (lys) ratios
(1:1, 1:2.5, 1:5, and 1:10). Mixed Anhydride (MA) Method.
Hapten 5 was conjugated to the same proteins at different
hapten:protein (lys) molar ratios (2:1, 1:1, 1:2.5, 1:5). In both
methods, 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 10 mM PBS at 1 mg
(2) Ch em ica ls a n d Im m u n och em ica ls. Immunochemicals
were obtained from Sigma Chemical Co. (St. Louis, MO).
Standards for cross-reactivity studies were purchased from
Aldrich Chemical Co. (Milwaukee, WI).
(3) Bu ffer s. PBS is 10 mM phosphate buffer, 0.8% saline
solution and unless otherwise indicated the pH is 7.5. Borate
buffer is 0.2 M boric acid-sodium borate, pH 8.7. Coating buffer
is 50 mM carbonate-bicarbonate buffer pH 9.6. PBST is PBS
with 0.05% Tween 20. Citrate buffer is a 40 mM solution of
sodium citrate pH 5.5. The substrate solution contains 0.01%
TMB (tetramethylbenzidine) and 0.004% H2O2 in citrate buffer.
(4) Non com p etitive Dir ect ELISA. The avidity of the
As53-As58 versus the enzyme tracers was determined by
measuring the binding of serial concentrations (1 to 0.015 µg
mL-1 in PBST, 100 µL/well) of each enzyme tracer (ET) to
microtiter plates coated overnight at 4 °C with 12 different
dilutions (1/1000 to 1/1024000 in coating buffer, 100 µL/well)
of each of As. The ET solutions were incubated in the coated
plates for 30 min at RT. The plates were then washed four times
with PBST, and the substrate solution (100 µL/well) was added
and incubated 30 min at room temperature before the enzymatic
reaction was stopped by adding 4 N H2SO4 (50 µL/well).
(5) Non com p etitive In d ir ect ELISA. The avidity of the
different As53-As58 for the coating antigens was determined
by measuring the binding of serial dilutions (from 1/1000 to
1/64000 in PBST; 100 µL/well) of each As to microtiter plates
coated overnight at 4°C with 12 different concentrations (from
10 to 0.005 µg mL-1 in coating buffer; 100 µL/well) of the 1-5
and 7-12 -BSA, -CONA, and -OVA conjugates. The As
solutions were incubated in the coated plates for 30 min at room
temperature. After that, the plates were washed four times with
mL-1
.
Ha p ten Den sity An a lysis. Hapten densities of the protein
conjugates were determined by matrix-assisted laser-desorption
ionization time-of-fly mass spectrometry (MALDI-TOF-MS) by
comparing the molecular weights of the unreacted proteins with
those of the conjugates. The MALDI-MS (matrix-assisted laser
desorption ionization mass spectrometer) used for analyzing the
protein conjugates was a Perspective Biosystems Time-of-Flight
(TOF) Mass Spectrometer Voyager-DE RP equipped with a laser
unit which operates with an intensity of 2800. The instrument
is controlled by a BioSpectrometry Workstation provided with
the software Voyager-DE-RP (version 4.03) developed by Per-
spective Biosystems Inc. (Framingham, MA) and GRAMS/386
(for Microsoft Windows, version 3.04, level III) developed by
Galactic Industries Corporation (Salem, NH). MALDI spectra
were obtained by mixing 2 µL of the matrix (trans-3,5-dimethoxy-
PBST, and
a solution of goat anti-rabbit IgG coupled to
horseradish peroxidase (antiIgG-HRP, 1/6000 in PBST) was
added to the wells (100 µL/well) and incubated for 30 min at
room temperature. The plates were washed again and the
substrate solution (100 µL/well) was added. Color development
was stopped after 30 min at RT with 4 N H2SO4 (50 µL/well)
and the absorbances were read at 450 nm. Optimal concentra-
tions for coating antigens and As dilution were chosen to
produce around 0.7 to 1 units of absorbance in 30 min.
(6) Com p etitive In d ir ect ELISA. Microtiter plates were
coated with 7-OVA (AE) (1:2.5) (0.6 µg mL-1 in coating buffer,

Biological Monitoring of 2,4,5-Trichlorophenol (I)
Chem. Res. Toxicol., Vol. 15, No. 11, 2002 1363
F igu r e 1. Stick display of the optimized geometries of 2,4,5-TCP and haptens A, B, and C according to MNDO models. Calculations
have been made using the corresponding amide derivatives to mimic the conjugated haptens. The elements are presented in the
following manner: light blue, carbon; dark blue, nitrogen; white, hydrogen; red, oxygen; yellow, chlorine.
aqueous media (ArOH f ArO- + H+). Thus, punctual
100 µl/well) overnight at 4 °C covered with adhesive plate
sealers. The following day the plates were washed with PBST
charges in the aromatic ring for both species (acid and
(five times, 300 µL/well). 2,4,5-TCP standards (400 to 0.04 nM,
prepared in PBS) or samples were added to the coated plates
(50 µL/well) followed by the sera As53 (1/1000 in PBST, 50 µL/
obtained for each atom of the ring and the sum of those
well). After 30 min of incubation at RT, a solution of anti IgG-
HRP (1/6000 in PBST) was added (100 µL/well) and the mixture
conjugated base) were calculated at their minimum
energetic conformations. Figure 2 shows the results
charges. It can be observed that there are almost no
differences in the punctual charges between the analyte
incubated for 30 min more at RT. The plates were processed as
and haptens A, B, and C when we do consider them as
organic acids. Only the balance of the total charge is
slightly more positive for hapten B (see Figure 2, left).
Quite the opposite, the punctual charges vary signifi-
cantly considering the haptens as conjugated bases. As
phenoxides, the charges of the target analyte and haptens
A and C follow the same pattern and the total charge is
more negative. In contrast, hapten B having the hydroxyl
group blocked cannot participate in this equilibrium,
keeping the same punctual distribution independently
from the pH of the media and the total charge remains
positive (see Figure 2, right).
Haptens A and C, thus, seem to better mimic the
analyte regarding both geometry and punctual charges
independently from a media with a pH higher or lower
than the respective pKa. Nevertheless, to apply these
considerations the tendency of these organic acids to give
protons to the media should be the same, that means all
three compounds must have similar pKa values. Accord-
ing to the Gibbs equation, on proton transfer reactions,
the deprotonation enthalpy (DPE) is directly related to
the pKa. Dewar et al. proved that semiempirical theoreti-
cal models provide reliable data regarding PA (proton
affinities) and DPE values if compared with experimental
data (52). Using MNDO semiempirical models, we have
been able to determine the formation enthalpies (∆H0)
of 2,4,5-TCP, hapten A and hapten C as organic acids
[Hfp(ArO-)] and as conjugated bases [Hfr(ArOH)]. Since,
described above.
(7) Sp ecificity Stu d ies. Stock solutions of different phenolic
compounds were prepared (1 mM in DMSO) and stored at 4
°C. Standard curves were prepared in PBS (10 000 nM to 0.04
nM) and each IC50 determined in the competitive experiment
described above. The cross-reactivity values were calculated
according to the following equation: (IC50 trichlorophenol/IC50
phenolic derivative) × 100.
Resu lts a n d Discu ssion
Ha p ten Design . The spacer arm necessary to link the
molecule to the protein could be introduced in three
different positions of the chemical structure of 2,4,5-TCP.
Figure 1 shows these chemical structures at their mini-
mum energetic levels after using MNDO models. The
introduction of the spacer arm does not produce signifi-
cant conformational changes since the three chemicals
conserve their planar geometries. Thus, in hapten A, the
spacer arm substitutes the hydrogen atom of the ortho
position. Although the linker is apparently too close to
the phenolic group, this hapten keeps the three chlorine
atoms at the same positions as the target analyte and
the phenolic group free. In hapten B, the aromatic ring
does not suffer any apparent geometric modification, and
the spacer arm is placed through the oxygen atom thus
maximizing the recognition of the aromatic group and
the particular distribution of the chlorine atoms. Finally,
hapten C respects the phenolic group by introducing the
spacer arm at the meta position of the aromatic ring.
As demonstrated with theoretical studies in our previ-
ous paper (44), the real differences between these haptens
and the target analyte are observed on the electronic
distribution of these molecules, especially considering the
acid-base equilibrium of the phenolic compounds in
∆H0 ) ∆Hfp - ∆Hfr
where ∆Hfr is the formation enthalpy of the reagents and
∆Hfp is the formation enthalpy of the products, the DPE
values of the different proton-transfer equilibria can be

1364 Chem. Res. Toxicol., Vol. 15, No. 11, 2002
Nichkova et al.
F igu r e 2. Total charge and punctual charge of 2,4,5-TCP and haptens A, B, and C as phenols (left) and phenoxides (right). The
relative amounts of these two forms depend on the corresponding pKa values (see Table 3). The bars show the total charge. Haptens
A and C mimic better the behavior of the target analyte in both situations. On top there are shown the acid-base equilibria and the
calculated deprotonation enthalpies (DPE) using MNDO semiempirical models and pKa values. The elements are presented in the
following manner: light blue, carbon; dark blue, nitrogen; white, hydrogen; red, oxygen; yellow, chlorine. For the graphs, the carbon
atoms have been numbered clockwise starting with the atom that supports the oxygen.
calculated according to the following equation:
and introducing the chlorine atoms in the last step (44).
However, the idea of introducing a chlorine atom at the
less active meta position of the aromatic ring seemed also
tricky. In contrast, the introduction of an alkylamino
group in ortho of 2,4,5-trichlorophenol, as it appears in
hapten A, had already been reported by Stokker et al.
for the preparation of a new class of saluretic agents (55).
Thus, following a similar procedure, we were able to
accomplish the preparation of 3-(2-hydroxy-3,5,6-tri-
chlorophenyl)propanoic acid (hapten A) with a moderate
yield (45, 49)
∆H0 ) [∆Hfp(H+) + ∆Hfp(ArO-)] -
∆Hfr(ArOH) ) DPE
According to the literature the formation enthalpy of the
H+ is 367.2 kcal/mol (52, 53). Figure 2 (top) shows the
DPE calculated for 2,4,5-TCP and haptens A and C
deprotonation equilibrium reactions using a MNDO
semiempirical models. The DPE values clearly indicate
the acidic character of these compounds, but the differ-
ences encountered between the calculated values did not
show large variations. This fact was additionally sup-
ported by theoretical calculations made for the pKa values
using a suitable software (see Figure 2, top). Although
hapten C and 2,4,5-TCP had a slightly closer pKa and
DPE values, overall we can conclude that both haptens
A and C mimic reasonably well the properties of the
target analyte.
From the synthetic point of view, the preparation of
hapten C introducing the spacer arm at the meta position
of the target analyte is more difficult. Regioselective
nucleophilic substitution at meta positions of phenolic
substances has been reported by preparing first arene-
tricarbonyl-chromium (0) complexes (54) of the corre-
sponding aromatic compound. Another strategy could go
beyond using 3(3-hydroxyphenyl)-propenoic acid as start-
ing material (possessing already the spacer arm in meta)
P olyclon a l An tiser a . Hapten A was coupled to KLH
and BSA following the mixed anhydride method (MA)
and the KLH conjugate was used to raise antibodies in
three white New Zealand rabbits. The obtained antisera
were named As53, As54, and As55. Antibodies against
hapten B (As56, As57, and As58) were already available
in our group. Thus, with the aim to prove the results of
the theoretical studies we decided to evaluate both groups
of antisera for their ability to bind 2,4,5-TCP on a
competitive ELISA.
Dir ect ELISA. Haptens A, 1-5, and 7-12 (see Table
1 for chemical structures) were coupled to HRP and
tested in a noncompetitive direct ELISA format. Recogni-
tion was observed only for the homologous or quasi-
homologous enzyme tracers. Thus, As53-As55 (A-KLH)
did only recognize A-HRP with an acceptable titer while
5-HRP was only recognized by As55. As56-As58 (B-

Biological Monitoring of 2,4,5-Trichlorophenol (I)
Chem. Res. Toxicol., Vol. 15, No. 11, 2002 1365
Ta ble 1. Ch em ica l Str u ctu r es of 2,4,5-TCP a n d P h en olic
Ha p ten s
Ta ble 3. Rela tive Sim ila r ities betw een th e Ta r get An a lyte
a n d th e Ha p ten s Rega r d in g P u n ctu a l Ch a r ge
Distr ibu tion
RMSE,c
pH 7.5
hapten
a
hapten
pKa
RO- (%)b
densityd
1
2
3
4
5
7
8
9
10
11
12
A
-
-
-
-
0.436
0.425
0.081
0.113
0.120
0.157
0.224
0.320
0.249
0.411
0.421
0.093
30
25
14
16
7
25
17
21
26
27
28
10
-
6.59
5.51
6.63
7.37
7.82
7.99
7.99
-
89.05
98.99
88.11
57.43
32.37
24.45
24.45
-
-
-
7.38
7.1
56.86
71.5
2,4,5-TCP
a
pKa values of the haptens are calculated in their amide form.
Percentage of the phenoxide species when the pH is 7.5. c The
b
similarity between the haptens and the analyte has been expressed
as the root-mean-square error (RMSE) of the differences between
the punctual charges of the corresponding atoms of both molecules
(hapten vs analyte) at pH 7.5. The punctual charge on each atom
was calculated considering the percentage of the neutral molecule
and their anion form at pH 7.5 according to their pKa values.
d
Hapten densities of the BSA conjugates measured by MALDI-
TOF-MS. The data corresponds to conjugates synthesized by the
active ester method, except for hapten 5 that corresponds to the
conjugate prepared by the mixed anhydride method. In both
methods the hapten:protein molar ratio employed during the
conjugation reaction 2:1 in terms of lysine residues of the protein.
These data has only been included as reference.
Ta ble 2. F ea tu r es of th e Com p etitive Dir ect ELISAs
The avidity of As53-As58 for the protein conjugates
was evaluated by two-dimensional checkerboard titration
experiments (data non shown). Similarly to the direct
ELISA, it was observed that As56-As58 (immunizing
hapten B) had lower avidity for the protein conjugates
than As53-As55 (immunizing hapten A). Taking into
account that the pH value of the assay buffer in these
experiments was 7.5, we can attribute this divergence
to the marked differences in the punctual charges of the
aromatic ring when the hydroxyl group is blocked (see
above, Figure 2). Thus, the competitors of phenyl-akyl
ether type, such as haptens 1, 2, 11, and 12, were the
best recognized by As56-As58. Quite the opposite and
as expected, As53-As55 showed higher avidity for the
haptens with the free phenolic group, especially if the
chlorine atoms were at the same positions as in the target
analyte. According to the pKa values of most of the
competitor haptens (see Table 3) an important fraction
of molecules are ionized at the assay pH. Only haptens
8, 9, and 10 had a pKa sufficiently high to remain mainly
in their protonated forms.
c
Asa
ETb
Amax
Amin
slope
IC50
r2
53
54
55
A-HRP
A-HRP
A-HRP
5-HRP
0.81
0.63
1.27
0.94
0
-0.43
-0.89
-1.03
-0.97
18.87
165.48
21.62
0.989
0.985
0.981
0.971
0.06
0.21
0.14
15.84
a
b
As, antisera. ET, enzyme tracer. c IC50 values are expressed
in µg L-1. Antisera 56-58 raised against hapten B-KLH did not
afford any competitive immunoassay.
KLH) did only recognize 2-HRP that only differs from
hapten B in the length of the spacer arm. However these
last As/enzyme tracer combinations produced only a
maximum absorbance of 0.3 units when the As was
diluted 1/500 times and the enzyme tracer was used at
a concentration of 2 µg mL-1. Thus, we focused on the
antisera raised against hapten A to test the ability of this
antiserum to bind 2,4,5-TCP on a competitive assay. All
the combinations gave competitive assays although the
detectability was too poor for our purposes. Table 2 shows
the features of these immunoassays. The best assay gave
a detectability of 16 µg L-1 in the middle point of the
assay. Therefore, we decided to explore the indirect
ELISA format with the aim to find a coating antigen/
antiserum combination with a better detectability. Al-
though the indirect format introduces an additional step
in the procedure, the absence of the enzyme during the
competition made it more promising regarding possible
matrix effects from the real biological samples (36).
In d ir ect ELISA. (1) Scr een in g of th e An tiser u m
Avid ity for th e Im m u n or ea gen ts. Haptens 1-4 and
7-12 were initially conjugated to BSA, OVA, and CONA
by the active ester (AE) method, and hapten 5 was
conjugated to the three proteins by the mixed anhydride
(MA) method. The hapten densities of the BSA conjugates
determined by MALDI-TOF-MS were quite high (see
Table 3).
(2) Com p etitive ELISA. Those As/coating antigen
combinations showing reasonable titers were used for
screening the ability of 2,4,5-TCP to inhibit binding of
the antibodies to the coated plates. In agreement with
the results from the molecular modeling studies, only
As53-55, raised against hapten A, were able to provide
competitive assays within the analyte concentration
interval investigated. Table 4 shows the features of the
immunoassays with IC50 values lower than 10 µg L-1
.
Almost all the assays have slope values close to 1 that
proves the high avidity of the antibodies for the target
analyte.
Using theoretical models, on a previous paper we
reported that the best immunoassay detectability was
accomplished using a competitor hapten with the highest

1366 Chem. Res. Toxicol., Vol. 15, No. 11, 2002
Nichkova et al.
Ta ble 4. Im m u n oa ssa y F ea tu r es of th e Com p etitive
In d ir ect ELISAa
Ta ble 5. Effect of th e Ha p ten Den sity of th e Com p etitor
on th e Im m u n oa ssa y F ea tu r esa
e
Asb
CAc
methodd Amax Amin
slope IC50
r2
hapten:
CA
proteinb
δc
Amax Amin slope IC50
r2
53 1-BSA
5-BSA
AE
MA
MA
AE
AE
AE
AE
AE
MA
MA
MA
AE
AE
AE
AE
AE
AE
AE
AE
0.61 0.03
1.02 0.08
0.68 0.03
0.94 0.06
0.75 0.01
0.70 0.02
0.86 0.01
0.57 0.13
-1.2
9.28 0.985
6.94 0.980
2.42 0.989
0.83 0.990
6.47 0.992
5.35 0.994
7.93 0.989
4.38 0.995
8.49 0.995
-0.63
-0.64
-1.2
5-BSA (MA)
2:1
1:1
1:2.5
1:5
2:1
7.5
9
5
1.02 0.08 -0.63 6.94 0.980
1.04 0.01 -0.46 1.30 0.980
0.92 0.01 -0.52 1.94 0.987
0.92 0.02 -0.45 1.56 0.989
5-OVA
7-OVA
8-OVA
-0.6
3
9-CONA
10-CONA
12-OVA
54 5-BSA
5-CONA
5-OVA
-0.96
-0.71
-0.86
-0.68
5-OVA (MA)
5-BSA (AE)
5-OVA (AE)
7-BSA (AE)f
n.d.e 0.68 0.03 -0.64 2.42 0.989
1:1
6
3
3
n.d.
0.82 0.01 -0.56 1.76 0.990
0.87 0.02 -0.54 1.92 0.990
1:2.5
1:5
1.3
0.21
0.61 0.01 -0.5
0.45 0.990
1.33 0.07
0.56 0.03
1.05 0.01
0.69 0.01
1.07 0.001 -0.5
0.63 0.01
0.98 0.2
0.82 0.02
1.01 0.04
0.73 0.04
-0.84 10.04 0.990
1:1d
1:2.5
1:5
-0.90
-0.78
-1.0
4.56 0.998
3.36 0.996
3.05 0.990
6.71 0.959
4.87 0.990
4.05 0.910
8.66 0.990
9.64 0.940
8.53 0.970
13
8
5
1.13 0.01 -0.48 15.52 0.981
7-BSA
7-OVA
8-BSA
0.8
1.1
0.01 -0.64 11.1 0.988
0.01 -0.56 9.13 0.989
1:10
1:1
1:2.5
1:5
1:10
2:1
1:1
1:2.5
1:5
1:10
2:1
1:1
1:2.5
1:5
20
12
6
1.19 0.01 -0.65 16.81 0.997
1.28 0.02 -0.72 9.70 0.980
1.24 0.01 -0.60 10.30 0.985
0.79 0.01 -0.68 4.80 0.988
10-OVA
55 1-BSA
2-BSA
-1.0
-0.96
-1.2
-1.3
-1.2
3
2-OVA
8-OVA
25
22
13
7
0.37 0.03 -1.69 0.24 0.964
0.59 0.01 -0.98 0.28 0.985
Only those assays reaching IC50 values below 10 µg L-1 are
a
0.3
0.1
0.01 -0.71 0.1 0.986
0.01 -1.19 0.06 0.919
shown in the table. As, antisera. c CA, coating antigen. Conju-
gation method used to prepare the coating antigens: AE is active
ester; MA is mixed anhydride. e IC50 values are expressed in µg
L-1. In bold are the best As/coating antigen combinations.
b
d
4
7-OVA (AE)
n.d. 0.94 0.06 -1.20 0.9 0.990
12
10
3
3
0.77 0.03 -1.24 0.57 0.990
0.72 0.01 -1.13 0.29 0.990
0.65 0.02 -1.00 0.21 0.990
0.74 0.02 -1.20 0.23 0.990
1:10
degree of heterology regarding punctual charge dis-
tribution and structural geometry (45). Persisting on the
aim to rationalize immunoassay development, we tried
also to provide objective data about the hapten heterolo-
gies and their correlation with the IC50 values of the
immunoassays obtained. Thus, all haptens 1-12 and the
target analyte were optimized to find their minimum
potential energies using a semi-empiric PM3 model. The
ratio phenol:phenoxide was calculated for each hapten
according to their pKa values and used to determine the
ultimate punctual charges of the molecule. The heterol-
ogy, as the difference between the charge distribution of
each hapten and the analyte, has been expressed as the
root-mean-square error (RMSE) of the addition of the
errors for each equivalent position of the aromatic rings
(competitor vs target analyte, see Table 3). With these
considerations the following heterology order according
to the electronic distribution on these molecules can be
defined: hapten 1 > hapten 2 g hapten 12 > hapten 11
. hapten 9 > hapten 10 > hapten 8 > hapten 7 > hapten
5 > hapten 4 > hapten A > hapten 3. The greatest
homology of hapten 3 in relation to hapten A and 2,4,5-
TCP can be explained considering the pKa values of these
substances. While hapten A has approximately only half
of the molecules ionized at pH 7.5, hapten 3 and 2,4,5-
TCP have 89 and 71%, respectively, in the ionized form.
However, it must be noticed that using hapten 3 as
immunizing hapten would had the risk of introducing
additional undesirable epitopes for antibody recognition.
According to these results, we can observe that the best
competitive assays were afforded by the competitors with
a high homology with the target analyte (see Table 4).
Except for the As55 in all other cases, haptens 5 and 7
afforded the most sensitive assays. Unfortunately, we did
not have sufficient hapten A to prepare coating antigens
by the AE method to prove if this was also true for this
competitor. However, in addition to the hapten homolo-
gies, other factors are also influencing the availability of
the competitor for the antibody interaction, when co-
valently forming part of the protein conjugate. Thus, the
hapten density or the protein nature, are also other
a
b
In all cases the antiserum used was As 53. Hapten:protein
molar ratio used for the conjugation reaction. The molar ratio of
the protein has been calculated in terms of the lysine residues
available. c Hapten densities (δ) are expressed as mol of hapten
per mol of protein, and the data reported has been measured by
d
MALDI-TOF-MS. 5-BSA (AE) (1:1) was not soluble. e nd, not
determined. f The conjugate with the highest density was not
recognized by the antibody.
aspects to consider. Similarly the hydrophobic or hydro-
philic nature of the compound may also determine its
tendency to be hidden or not inside the tertiary structure
of the protein.
ELISA Op tim iza tion a n d Ch a r a cter iza tion . Im-
munoassays 5-OVA/As53 and 7-OVA/As53 were selected
for further optimization and evaluation because of their
detectability, good signal-to-noise ratio, acceptable slope,
and reproducibility (see Table 4). The initial studies were
aimed to improve immunoassay detectability. It has been
reported that the degree of hapten density of the competi-
tors can affect the immunoassay sensitivity (44, 56, 57).
The carrier protein OVA has 20 lysine residues, most of
which are available for hapten coupling (58). In this case,
the hapten densities of the corresponding 5-BSA and
7-BSA conjugates measured by MALDI-TOF-MS were 7
and 25 mol/mol of protein (see Table 3). The difference
encountered between those two conjugates can be ex-
plained by the fact that hapten 5 had been conjugated
by the MA method, which usually gives lower yields. To
study the effect of the hapten density, hapten 5 was
conjugated again to BSA and OVA by both methods (MA
and AE) and hapten 7 by the AE method at different
hapten:lys residues molar ratios. The resulting degree
of conjugation was determined by MALDI-TOF-MS (see
Table 5). For the case of hapten 5, the assays reached
lower IC50 values (higher detectability) when the MA was
the conjugation method used. In general for both haptens,
it was observed that, the lower the hapten density, the
higher the immunoassay detectability (lower IC50 values).
The same effect has been observed on other immuno-
assays such as sulfamerazine (56), chlorpyrifos (57), or

Biological Monitoring of 2,4,5-Trichlorophenol (I)
Chem. Res. Toxicol., Vol. 15, No. 11, 2002 1367
as compromise because of the greater Amax/IC50 ratio
encountered at this time (see Figure 3).
Finally, we must mention that the concentration of
Tween 20 did not significantly influence the features of
the 2,4,5-TCP immunoassay. Only when the concentra-
tion reached values of 0.5 and 2.5% a significant increase
of the IC50 was observed, achieving values close to 6 µg
L-1 without affecting Amax. This result is in agreement
with the idea suggested by some authors that polar
analytes are less influenced by the presence of detergent
because of their inability to establish nonhydrophobic
interactions with it (45, 46, 49, 60). Next experiments
were performed at 0.025% Tween 20 final concentration
by preparing the standards in PBS and the As in PBST
(0.05% Tween 20).
F igu r e 3. Variation of the immunoassay parameters (IC50 and
Amax) as function of the length of the competitive step. The
analyte, 2,4,5-TCP and the antibody were incubated at different
times (between 10 and 120 min) in the antigen-coated plates.
The results reported are extracted from the four-parameter
equation used to fit the standard curves. Each standard curve
was run in duplicate.
As result of this evaluation we obtained an assay using
As53 and 7-OVA (molar ratio 1:2.5, hapten density 10
mol of hapten/mol of protein) as coating antigen. The
assay did not introduce any preincubation of the As with
the analyte and had a competitive step of 20 min. The
assay buffer was 10 mM PBS, and the As was diluted in
the same buffer containing 0.05% Tween 20 (final con-
centration in the assay 0.025%). Using these conditions,
the assay showed an IC50 of 0.61 µg L-1 and a LOD of
0.084 µg L-1 (see Figure 4).
2,4,6-TCP (44). This is consistent with the statement that
the least hapten centers are available in the coating
antigen, the least amount of analyte in solution is needed
to compete for the limited number of antibody binding
sites. The detectability improved significantly by dimin-
ishing the hapten density for the As53/7-OVA assays.
Finally, the coating antigen 7-OVA (AE 1:2.5 molar ratio)
was selected for further studies, since lower molar ratios
did not increase significantly the immunoassay detect-
ability, and it was necessary to employ higher concentra-
tions of immunoreagents to obtain the same maximum
absorbance in the assay (see Table 5).
Although it has been reported that the sensitivity of
an immunoassay can be improved by preincubating of
the analyte with the As prior to the competition (43, 59),
in this case no significant effect on the detectability was
observed after incubating the As with the analyte over-
night at 4 °C (IC50 0.29 µg L-1 versus 0.31 µg L-1). The
same effect was observed for other ELISAs developed in
our group for phenolic compounds (44-46). On the other
hand, shortening the length of the competitive step
improved the immunoassay detectability (the lowest
IC50). Thus, an increase of the IC50 value of three times
was observed when varying the incubation length from
10 min to 2 h. However, a concomitant increase of the
maximum assay signal (Amax) was also produced. There-
fore, a competitive incubation time of 20 min was chosen
Immunoassay specificity was evaluated by testing 22
structurally related compounds as competitors. The
results of the crossreactivity studies shown in Table 6
demonstrate that the assay is quite specific. It can be
observed that the degree of recognition was directly
related to the presence of two chlorine atoms (one at ortho
and another at para position) and the hydrogen in meta
as in the target analyte. For example, 2,3,4,6-tetrachloro-
phenol (TtCP) possessing these three atoms on one of its
moieties was the most recognized compound with 19%
crossreactivity value, while 2,3,4,5-TtCP lacking the
hydrogen atom in meta was less recognized (4.6%) and
2,3,5,6-TtCP lacking the chlorine atom at para position
and possessing two chlorine atoms at meta positions
cross-reacted only 0.5%. Similarly, among the trichloro-
phenols 2,4,6-TCP is the most recognized (17%) possess-
ing both ortho and para chlorine atoms and a meta
hydrogen, in the same range as 2,3,4,6-TtCP. The lack
of the hydrogen in meta (2,3,4-TCP) or of the chlorine
atom in para (2,3,5-TCP and 2,3,6-TCP) decreases sig-
nificantly the recognition. Analytes with only two, one
or none chlorine atoms had a negligible (e3.5%) or void
interference in the assay. As reported (44, 45, 49),
F igu r e 4. Calibration curve of the 2,4,5-TCP immunoassay using As53 and 7-OVA (hapten density, 10). The assay is performed in
10 mM PBST (0.025% Tween 20). The curves were run in duplicate. On the right side of the graph are shown the features of this
immunoassay.

1368 Chem. Res. Toxicol., Vol. 15, No. 11, 2002
Nichkova et al.
Ta ble 6. In ter fer en ce Ca u sed by Str u ctu r a lly Rela ted
Ch em ica ls, Exp r essed by Th eir IC50 a n d th e P er cen ta ge
of Cr ossr ea ctivitya
seem to indicate that for this type of analyte the punctual
charge distribution of the molecule is one of the most
important parameters.
phenolic
compds
IC50
(nM)
%
CR
brominated IC50
analogues (nM)
%
CR
On the other hand a degree of heterology has been
established for a battery of 13 different haptens (includ-
ing the immunizing hapten) used as competitors accord-
ing to their punctual charge distribution calculated at
their minimum energetic level and considering the pKa
value of these substances (see Table 3). By correlating
this parameter with the detectability afforded by the
different assays obtained, we have arrived to the conclu-
sion that the best performance occurs when the competi-
tor has a moderate-to-high degree of homology. However,
this result should not be taken as a general rule since
other factors determining the way the competitor haptens
are available for antibody recognition also play an
important role. Thus, in this aspect we must notice that
the work presented here proves once more that detect-
ability improves with competitors showing a moderate
hapten density.
The indirect ELISA reported here shows a limit of
detection of 0.086 µg L-1 and it is quite specific for 2,4,5-
TCP. As occurred with other reported assays (44, 45, 49)
the brominated analogues may also be recognized. The
detectability accomplished by this immunoassay in the
assay buffer is sufficient for environmental and biological
monitoring studies. As mentioned in the Introduction, the
concentrations found in water and urine samples are
often above the limit of detection (0.086 µg L-1). Further
work has been focused on the evaluation of this assay to
analyze chlorophenols in body fluids (61).
no.b
5
4
PCP
2,3,4,6-TtCP
2,3,4,5-TtCP 33
2,3,5,6-TtCP 304
101.3
8
1.5
PBP
361.9
0.42
18.9
4.6
0.5
100
17
0.9
0.1
0.4
3.5
0.9
0.45
3
2
2,4,5-TCP
2,4,6-TCP
2,3,4-TCP
2,3,5-TCP
2,3,6-TCP
2,4-DCP
2,5-DCP
2,6-DCP
3,4-DCP
2-CP
1.52
8.9
168.9
1520
380
43.4
168.9
337.8
2,4,6-TBP
5.5 27.7
2-B-4-CP
2,4-DBP
2,6-DBP
14.9 10.2
14.5 10.5
108.6
1.4
>10000 <0.01
2533.3 0.06
>10000 <0.01
1
3-CP
4-CP
>10000 <0.01 4-BP
475
0.32
a
Crossreactivity is expressed as % of the IC50 of 2,4,5-TCP/IC50
b
phenolic compound. Number of halogens. B, bromo; C, is chloro;
DCP, dichlorophenol; TCP, trichlorophenol; TtCP, tetrachloro-
phenol; PCP, pentachlorophenol; BP, bromophenol; DBP, dibromo-
phenol; TBP, tribromophenol; PBP, pentabromophenol.
brominated phenols were also highly recognized in this
assay. Thus, 2,4-dibromophenol (DBP) cross-reacted 10.5%
while its homologous chlorinated analyte 2,4-DCP was
only recognized 3.5%. Similarly, 2,4,6-tribromophenol
(TBP) was recognized 27.7% while 2,4,6-TCP interfered
only 17%. Unfortunately, at the time of writing this
paper, we have not been able to test 2,4,5-TBP, but we
would expect recognition greater than 100%.
Ack n ow led gm en t . This work has been supported
by the EC Program (Contract QLRT-2000-01670) and
by CICYT (BIO2000-0351-P4-05 and ALI99-1379-CE).
Mikaela Nichkova thanks the Spanish Ministry of Sci-
ence for her fellowship of the FPU Program.
Con clu sion s
An immunoassay has been developed for the detection
of 2,4,5-TCP making use of molecular modeling tools to
assist on the hapten design of the chemical structures of
the immunogen and the competitors. The theoretical
studies have been useful to rationalize certain aspects
related to the avidity of the different antisera obtained.
Regarding the chemical structure of the immunizing
hapten, the antibodies raised against hapten B did not
afford any usable assay, while several immunoassays
were obtained with the antisera raised against hapten
A. From the synthetic point of view hapten B was easy
to prepare however the theoretical studies provide ra-
tional criteria to understand the lack of avidity of As56-
As58 for the target analyte. Thus, 2,4,5-TCP, hapten A
and hapten C are organic acids that in the aqueous media
of the assay buffer are in equilibrium with their corre-
sponding phenoxide forms. These two forms differ con-
siderably on their punctual charge distribution. It is
therefore very important to know their actual ratio in
the assay conditions (see Figure 2). However, the absence
of a free hydroxyl group in hapten B prevents it from
behaving as an organic acid, and its punctual charge
distribution is more close to the protonated form of the
analyte than to the corresponding conjugated base. Thus,
at pH 7.5, 71% of the molecules of 2,4,5-TCP are ionized
and similarly occurs with haptens A and C. Therefore,
the higher frequency of the anionic forms and the strong
differences encountered in the punctual charges with the
neutral form of hapten B explains the lack of recognition
showed by these antibodies. In general, the results
obtained are in agreement with previous studies (44) and
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