Synthesis of site-heterologous haptens for high-affinity anti-pyraclostrobin antibody generation

Article abstract of DOI:10.1039/c0ob00686f

The design and synthesis of functional chemical derivatives of small organic molecules is usually a key step for the intricate production of a variety of bioconjugates. In this respect, the derivatization site at which the spacer arm is introduced in immunizing conjugates constitutes a highly critical parameter for the generation of high-affinity and selective antibodies. However, due to the usual complexity of the required synthetic procedures, the appropriate comparison of alternative tethering positions has often been neglected. In the present study, meticulous strategies were followed to prepare synthetic derivatives of pyraclostrobin with the same linkers located at diverse rationally-chosen sites. Activity appraisal of antibodies and bioconjugates was carried out by bidimensional competitive direct and indirect immunoassays, and a superior performance of two of the three synthesized haptens was found. Finally, a detailed analysis of the conformations of the target molecule and the synthesized haptens in aqueous solution was done using computer assisted molecular modeling techniques. This study suggested that the lower titers and affinities of one set of antibodies are most probably due to conformational effects of the spacer arm in the immunizing bioconjugate. The Royal Society of Chemistry 2011.

Full text of DOI:10.1039/c0ob00686f

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PAPER
Synthesis of site-heterologous haptens for high-affinity anti-pyraclostrobin
antibody generation†
Josep V. Mercader,a Consuelo Agullo´,b Antonio Abad-Somovilla*b and Antonio Abad-Fuentes*a
Received 7th September 2010, Accepted 22nd November 2010
DOI: 10.1039/c0ob00686f
The design and synthesis of functional chemical derivatives of small organic molecules is usually a key
step for the intricate production of a variety of bioconjugates. In this respect, the derivatization site at
which the spacer arm is introduced in immunizing conjugates constitutes a highly critical parameter for
the generation of high-affinity and selective antibodies. However, due to the usual complexity of the
required synthetic procedures, the appropriate comparison of alternative tethering positions has often
been neglected. In the present study, meticulous strategies were followed to prepare synthetic derivatives
of pyraclostrobin with the same linkers located at diverse rationally-chosen sites. Activity appraisal of
antibodies and bioconjugates was carried out by bidimensional competitive direct and indirect
immunoassays, and a superior performance of two of the three synthesized haptens was found. Finally,
a detailed analysis of the conformations of the target molecule and the synthesized haptens in aqueous
solution was done using computer assisted molecular modeling techniques. This study suggested that
the lower titers and affinities of one set of antibodies are most probably due to conformational effects of
the spacer arm in the immunizing bioconjugate.
thus facilitating its recognition by the receptor despite the fact that
direct coupling of the analyte to the carrier protein, when possible,
Introduction
has occasionally been employed.2 Although the physico-chemical
characteristics of the analyte determine the optimum properties
of the spacer arm for hapten conjugation, linear aliphatic bridges
are known to cause negligible interferences over the production
of antibodies if an optimum length of two to six carbon atoms is
maintained.3 Nevertheless, a critical parameter for the production
of antibodies is, most noteworthy, the derivatization site; that is,
the position at which the spacer arm is attached to the molecule.
The linker position will mold the final conformation of the
conjugate and therefore it will settle the specific moieties that will
be accessible for binding. Even if these general considerations
are simple and qualitative, they have generally been extremely
useful—in combination with rational and judicious chemical
examination of the target molecule by qualified chemists—for the
synthesis of appropriate haptens and bioconjugates applied to
the production of antibodies with the desired binding properties.
Lately, molecular modeling techniques are being increasingly used
as complementary tools to assist researchers in, for instance, the
prediction of the best hapten structures for immunoconjugate
preparation or the interpretation of experimental results regarding
affinity or selectivity of receptors.4
The production of high-affinity and selective binders to small
organic molecules is a biotechnological research field of huge
importance for such diverse disciplines as drug discovery, clinical
diagnostics, food safety, and environmental monitoring. Antibod-
ies are the paradigm of binding proteins due to their exquisite
molecular recognition ability. Long ago, Nobel-prize winner Karl
Landsteiner was the first to discover that low molecular weight
compounds become immunogenic only after association with a
carrier protein, coining the term hapten for these substances.1
Because small organic molecules usually lack ready-to-activate
chemical groups for protein conjugation, the generation of high-
quality antibodies exhibiting the desired ligand-binding features
strongly relies on the synthesis of suitable functional derivatives of
the target analyte. Such synthetic derivatives should closely mimic
the target ligand, maximizing the steric, hydrophobic, and elec-
tronic similarity to the parent molecule. Most commonly, synthetic
haptens incorporate a spacer arm to better display the molecule
aDepartment of Organic Chemistry, Universitat de Vale`ncia, Doctor Mo-
liner 50, 46100, Burjassot, Vale`ncia, Spain. E-mail: antonio.abad@uv.es;
Fax: +34-963544328; Tel: +34-963544509
bDepartment of Biotechnology, IATA-CSIC, Agust´ı Escardino 7, 46980, Pa-
terna, Vale`ncia, Spain. E-mail: aabad@iata.csic.es; Fax: +34-963636301;
Tel: +34-963636301
Strobilurins are a new class of synthetic biocides displaying
outstanding properties and a new mode of action, so the introduc-
tion of the first strobilurin fungicides in 1996 meant a significant
contribution to the fight against fungal diseases.5 As part of an
ongoing project aimed at obtaining high-affinity antibodies and
developing immunochemical methods to selectively detect the
† Electronic supplementary information (ESI) available. Full character-
ization data of all the intermediate compounds in the synthesis of
haptens PYa6 and PYs5 are given as well as complementary data about
conformational studies and copies of 1H NMR and MS spectra. See DOI:
10.1039/c0ob00686f
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most relevant chemicals of the strobilurin family of pesticides,
we chose pyraclostrobin (PY) as a model analyte to ascertain
the influence of the spacer arm attachment site on the affinity of
the derived antibodies. PY is considered as a second generation
strobilurin fungicide, and it is composed of a methoxycarbamate
toxophore moiety, an aryl bridge, and a characteristic two-ring
system (Fig. 1). In this article, we describe the synthesis of PY
derivatives with the spacer arm located at different sites of the
molecule. To rigorously compare the suitability of these synthetic
haptens for inducing the formation of high-affinity antibodies, a
homogenous linker of comparable length and composition was
used for coupling to the same carrier protein. Very few examples
have been found in the literature in which the same spacer arm was
used to prepare several functionalized haptens through different
derivatization sites in order to find the optimum orientation of
the molecule.6 Antisera, representing the unbiased whole-response
of the animal immune system, were chosen as the source of
antibodies. The affinity and selectivity of the generated reagents
were evaluated by competitive enzyme-linked immunosorbent
assay (cELISA) using the conjugate-coated indirect and the
antibody-coated direct formats. Finally, our results were assessed
with computer-generated molecular models of the target molecule
and the synthesized haptens.
Results and discussion
Chemistry
After a rational examination of the planar structure of PY
(Fig. 1), three key positions for the introduction of the spacer
arm and analyte functionalization became readily apparent: the
methoxycarbamate toxophore moiety, the arylic bridge, and the
chlorophenyl moiety. Accordingly, three PY analogues, namely,
PYo5, PYa6, and PYs5 (Fig. 1), were proposed as haptens for
the production of high-affinity antibodies. All three structures
maintained the complete hydrocarbon skeleton and the most
characteristic functional groups of the analyte, incorporating the
hydrocarbon spacer arm at positions where minimum modifi-
cations of steric and electronic properties of the molecule were
expected. Moreover, the length and composition of the spacer
arms were nearly identical in all three haptens. The synthesis
of haptens PYa6 and PYs5 is herein reported for the first time,
whereas the preparation of hapten PYo5 was previously described
by our group in a study dealing with the influence of the
spacer arm length on antibody production.7 In hapten PYa6, the
spacer arm is directly attached to the aryl ring that contains the
methoxycarbamate group through a single carbon-carbon bond,
while in hapten PYs5 the hydrocarbon spacer chain is bonded
to the other (opposite) aryl group through a sulfur bridge that
replaced the chlorine atom of PY. A preliminary examination
of the synthetic routes required for the preparation of haptens
PYa6 and PYs5 evidenced that rather laborious and multi-step
schemes would be required to prepare these analogues. Initial
attempts to introduce the spacer arm at the required positions
directly on PY failed, so haptens had to be finally prepared
by total synthesis starting from ready available materials. We
used an adaptation of the strategy previously reported for the
preparation of PY8 and related compounds,9 which involved, for
such cases, the alkylation of a 1-aryl-1H-pyrazol-3-ol with a methyl
2-(bromomethyl)-phenyl(methoxy)carbamate as the key synthetic
step for the elaboration of the characteristic PY framework.
The synthesis of hapten PYa6 (11) began with the iodination
reaction of methyl methoxy(o-tolyl)carbamate (1) using iodine and
silver sulfate in AcOH (Scheme 1). This reaction is highly efficient
Fig. 1 Structure of PY and the three synthetic haptens.
Scheme 1 Synthesis of hapten PYa6.
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Table 1 Molar ratios of the different bioconjugatesa
and regioselective, affording exclusively the 4-iodo compound 2.
Radical benzylic bromination of 2 with N-bromosuccinimide and
azobisisobutyronitrile as the initiator under standard thermal
conditions afforded a very low yield of the desired benzylic
bromide, a result that contrasted with that obtained in the
bromination reaction of the non iodinated analogue (see below).
However, an acceptable yield of benzyl bromide 3 was obtained
when the free radical bromination of iodide 2 was undertaken with
bromine under radiation-induced conditions at room temperature.
Under these conditions, the reaction afforded a mixture of
bromide 3 and unreacted starting material, together with minor
amounts of the corresponding benzylic dibrominated derivative,
which could not be separated by conventional chromatographic
methods, so it was used as such in the following step. Thus,
the expected benzyl ether 7 was obtained in excellent yield from
the reaction of this mixture with 1-(4-chlorophenyl)-1H-pyrazol-
3-ol (6), readily prepared according to a literature procedure10
by reaction of (4-chlorophenyl)hydrazine hydrochloride (4) with
methyl acrylate and oxidation of the resulting pyrazolidinone
5 with oxygen and catalytic CuCl in about 57% overall yield.
Once the tricyclic ring system present in PY had been prepared,
the synthesis of hapten PYa6 was completed via substitution
of the iodine atom in 7 by the C6 hydrocarbon chain using
palladium catalyzed coupling methodology. Thus, Sonogashira
cross-coupling of iodide 7 with alkyne tert-butyl ester 8 under
standard conditions afforded the acetylenic compound 9, which
was hydrogenated using Wilkinson’s catalyst to give compound
10 in 90% overall yield for the two steps. The synthesis of hapten
PYa6 was completed by hydrolysis of the tert-butyl ester group of
10, which took place in nearly quantitative yield by treatment with
trifluoroacetic acid at 0 ◦C.
BSA
OVA
HRP
Abs.
Hapten
Abs.b
MALDIc
Abs.
PYo5
PYa6
PYs5
19
25
17
12
15
17
5
8
6
4
6
3
a Hapten densities were calculated as moles of hapten per mole of protein.
b Hapten densities retrieved by differential absorbance measurements.
c Hapten densities calculated by MALDI-TOF-MS.
sulfide chain was efficiently carried out by Cu-catalyzed cross-
coupling reaction of the aryl iodide with the thiol 16, which
was in turn prepared from tert-butyl 5-bromopentanoate (15)
and thiourea in very high yield via formation of the thiouronium
salt followed by cleavage with sodium pyrosulfite.11 The synthesis
of the whole framework of hapten PYs5 was completed by
alkylation of the pyrazolol moiety of 14 with the known methyl 2-
(bromomethyl)phenyl(methoxy)carbamate (18) in a similar way to
that described above for the other hapten. Under these conditions,
the alkylation reaction afforded the tert-butyl ester 19 in an
excellent 89% yield. Finally, acid hydrolysis of the tert-butyl ester
group of 19 by trifluoroacetic acid treatment gave the desired
hapten PYs5 (20) with also an excellent yield.
Bioconjugates and antibodies
The hapten density of the immunogenic conjugates is a funda-
mental parameter that determines the titer and the affinity of the
generated antibodies. Hapten’s carboxyl groups were activated for
coupling to the e-amine residues of the carrier proteins by standard
procedures (see experimental part). The hapten-to-protein molar
ratios (MR) of the three bioconjugates to bovine serum albumin
(BSA) were determined by differential absorbance measurements
and MALDI-TOF-MS. Similar values were retrieved by both
analytical methods (Table 1) as also observed by other authors,12
except for the MR of BSA–PYa6 when calculated by differ-
ential absorption, which seemed to be overestimated. Anyhow,
The synthesis of hapten PYs5 (20) began with the prepara-
tion of 1-(4-iodophenyl)-1H-pyrazol-3-ol (14) (Scheme 2), which
was obtained from (4-iodophenyl)hydrazine (12) via a two step
process involving condensation with tert-butyl acrylate catalyzed
by potassium tert-butoxide and subsequent oxidation of the
obtained dihydropyrazolone 13 with air catalyzed by CuCl.
The pyrazolol 14 was obtained with an overall yield of about
60%. Replacement of the iodine atom in 14 by the alkyl
Scheme 2 Synthesis of hapten PYs5.
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Table 2 Assay parameters achieved with homologous conjugates in the two cELISA formats
Indirect cELISAa
Direct cELISAb
Antiserum
Antiserum dilution (¥103)
Amax
IC50
[Tracer] (ng mL-1)
Amax
IC50
rPYo5#1
rPYo5#2
rPYa6#1
rPYa6#2
rPYs5#1
rPYs5#2
30
30
3
1.40 0.29
1.24 0.14
0.90 0.25
1.19 0.01
0.92 0.33
0.85 0.09
4.9 2.0
6.8 1.8
14.4 4.5
43.0 5.9
5.0 1.0
1.7 0.8
300
100
300
300
30
0.94 0.09
0.93 0.26
0.87 0.01
0.46 0.07d
1.38 0.18
0.86 0.12
10.2 0.9
8.4 2.6
> 100
> 100
6.9 1.1
1.5 0.3
10c
10
10
10
a Coating with homologous conjugate at 100 ng mL-1. b Antibody dilution was 1/3000. c Assay obtained with 1.0 mg mL-1 homologous conjugate. d Amax
above 0.5 could not be reached.
equivalent MRs for the three immunogens were found as required
for a proper comparison of the immune response. The observed
MRs of our BSA conjugates were in the same range as the
optimum MRs for immunogenic conjugates determined by Boro
et al.13 using the same carrier protein. For assay conjugates,
carrier proteins different to the immunogen are demanded,
so ovalbumin (OVA) and horseradish peroxidase (HRP) were
employed. Considering the calculated MRs of these conjugates,
it could also be seen that both of the PYa6 assay conjugates were
slightly higher than those of the other haptens, as it happened
with the BSA–PYa6 conjugate. Unfortunately, the MRs of OVA
conjugates could not be determined by MALDI-TOF-MS, and
the same mass spectra were obtained for the unconjugated and
the conjugated HRP samples, probably due to the low density of
hapten molecules in the conjugates. In any case, all assay (HRP
and OVA) conjugates behaved correctly in the cELISA at usual
concentrations, demonstrating that they carried sufficient hapten
molecules for adequate assay performance.
better (lower IC50 values) when OVA–hapten conjugates were
employed at 0.1 mg mL-1 (Table 2). Under optimum conditions
for each combination of immunoreagents, antibodies derived from
haptens PYo5 and PYs5 recognized PY with a similar affinity, with
antiserum rPYs5#2 displaying the lowest IC50 value (1.7 nM). On
the contrary, antisera from hapten PYa6 showed lower affinity.
These divergent results were even more evident in the antibody-
coated direct format, in which antibodies from hapten PYa6 clearly
did not perform as advantageously as the antibodies derived from
the two other haptens. Fig. 2 shows the standard curve for the
optimized assay in the antibody-coated direct cELISA format
resulting from the combination of antiserum rPYs5#2 with its
homologous tracer HRP–PYs5. This assay afforded a theoretical
limit of detection of 0.13 nM (50 ng L-1).
Six rabbits were immunized and blood samples were collected
after the third immunization. Titers of the two BSA–PYa6
immunized animals were approximately 104, whereas those of
animals immunized with BSA–PYs5 and BSA–PYo5 were around
10 and 50 times higher, respectively. The titers did not improve
after the fourth immunization, and consequently the animals were
exsanguinated. Finally, two antisera were generated from each
immunogen: antisera rPYa6#1 and #2 from immunogen BSA–
PYa6, antisera rPYo5#1 and #2 from immunogen BSA–PYo5,
and antisera rPYs5#1 and #2 from immunogen BSA–PYs5.
The influence of the derivatization site of assay conjugates
was evaluated for the two most common cELISA formats.
Depending on the ELISA format, OVA–hapten conjugates were
used as passive immobilized competitive reagent for indirect
assays whereas HRP–hapten conjugates were employed as enzyme
tracer conjugates in direct assays. The binding properties of the
generated polyclonal antibodies were initially evaluated as the IC50
value of the inhibition curve that was retrieved in homologous
competitive assays with PY as free analyte (Table 2). Standard
curves were run using an array of concentrations of the antibody
and the assay conjugate, so that a family of inhibition curves was
generated; one curve for each combination of immunoreagents.
This approach enabled a simultaneous, and therefore comparable,
determination of the IC50 values under different experimental
conditions, so information about antibody affinity was rapidly
acquired without a previous titration assay. We found that, in the
conjugate-coated indirect format, the antisera generally performed
Fig. 2 Standard curve for the optimized direct cELISA using antiserum
rPYs5#2 and the homologous tracer. The Amax value was 0.84. Values are
the mean of three independent experiments.
The antibody apparent affinity that results from the calculated
IC50 values may be influenced by the hapten conjugate that is used
for solid phase coating in indirect assays or that is coupled to the
enzyme in direct assays. Therefore, cELISAs were also undertaken
with heterologous conjugates for each antiserum (Table 3). Both,
PYs5-type antisera and PYo5-type antisera recognized the coating
OVA–PYa6 conjugate. However, very modest improvements of the
IC50 values, if any, were observed with these heterologous assays
as compared with the corresponding homologous combinations.
On the contrary, PYa6-type antisera did increase their apparent
affinity to PY with heterologous OVA conjugates. As expected, the
antibody-coated direct format was much more demanding about
using heterologous haptens because no binding (or not enough
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Table 3 Assay parameters achieved with heterologous conjugates in the two cELISA formatsa
Indirect cELISAb
Direct cELISAc
IC50 (nM) HRP conjugate [Tracer] (ng mL-1) Amax
Antiserum OVA conjugate
Antiserum dilution (¥103) Amax
IC50 (nM)
rPYo5#1
rPYo5#2
rPYa6#1
rPYa6#2
rPYs5#1
rPYs5#2
PYa6
PYa6
PYo5d
PYo5
PYa6
30
10
3
30
3
1.23 0.22 2.4 0.3
PYa6
PYa6
300
300
1.01 0.10 6.3 0.5
0.91 0.22 22.2 6.1
1.57 0.14 6.2 1.4
1.01 0.32 7.5 4.0
0.95 0.09 15.4 1.9
1.28 0.32 4.2 0.9
a Only results of those immunoreagent combinations that afforded Amax values over 0.5 are given. b Coating conjugate was at 1.0 mg mL-1. c Antibody
dilution was 1/3000. d Coating conjugate concentration was 0.1 mg mL-1.
binding) to the tracer conjugate was observed in most cases. As a
matter of fact, heterologous direct assays could only be developed
with PYo5-derived antibodies in combination with the enzyme
tracer HRP–PYa6 (Table 3). Interestingly, antiserum rPYs5#2,
displaying the highest affinity to PY in the homologous assays, was
the only antibody unable to recognize any heterologous haptens.
Overall, the same conclusion was deduced from homologous and
heterologous assays; that is, PYo5 and PYs5 haptens performed
better as immunogens than hapten PYa6.
For selectivity studies, competitive assays were run in the
conjugate-coated indirect format with all of the antibodies at
the selected immunoreagent concentrations to give Amax values
between 1.0 and 1.5 and using PY and other analytes as
competitors. Homologous conjugates were used in each case
at 0.1 mg mL-1, except for antiserum rPYa6#2, for which the
Fig. 3 Two views of the lowest energy conformer found for PY observed
OVA–PYa6 conjugate was used at 1.0 mg mL-1. Remarkably, no
from two different points of view. In the upper structure, the elements are
member of the strobilurin family other than PY was recognized
by any of the antisera, disregarding the derivatization site of
the immunogen. All of the strobilurin fungicides (azoxystrobin,
picoxystrobin, kresoxim-methyl, trifloxystrobin, dimoxystrobin,
fluoxastrobin, and orysastrobin) were assayed up to 1 mM. The
selectivity of the six antisera towards other fungicides like boscalid,
famoxadone, fenamidone, or cyazofamid, which are commonly
formulated together with PY, was also investigated. Again, none
of the antisera bound any of these compounds.
represented in the following manner: carbon, grey; oxygen, red; nitrogen,
blue; and chlorine, green. The lower structure represents the electron
density surface colored by electrostatic potential of a different view of
the most stable conformation of PY. The energy values [in atomic units
(au)] at each color interface are: white–red, +0.09 au; red–yellow, +0.02
au; yellow–green, +0.01 au; green–light blue, 0.00 au; light blue–dark blue,
-0.01 au; dark blue–pink, -0.03 au; pink–violet, -0.06 au; where 1 au =
627.503 kcal mol-1.
and the other plane was formed by the ortho-substituted benzene
ring—with an angle between them varying from 100◦ to 120◦. A
more accurate representation of the true shape of PY is given by the
electron density isosurface showed in the bottom of Fig. 3 which
also reflects the electrostatic potential at every point on the surface.
Electronic distribution and geometry are objective criteria to assist
hapten design and interpret antibody behavior. Our experimental
data clearly evidenced the advantage of haptens PYo5 and PYs5
over PYa6 with regard to the production of anti-PY antibodies
(Tables 2 and 3). Sanvicens et al.15 found that the best antibody
for trichloroanisol was obtained with an immunizing hapten
in which the spacer arm had been introduced by substitution
of a C–Cl bond by a C–C linkage, which made the aromatic
carbon atom much more positive. In contrast, the electronic
distribution played a major role in the production of antibodies
against trichlorophenol.6b To gain some insights into the molecular
reasons underlying our results, the electronic properties of the
three haptens were simulated by attaching the linkers to the
appropriate position of the lowest-energy PY conformer. As
depicted in Fig. 4, the introduction of the spacer arm did not
cause a significant alteration of the atomic charges, and very close
Computational analysis
PY is a medium-sized hapten with a very high degree of conforma-
tional flexibility due to the large number of bonds that display free
rotation (see Table 1SI in the ESI†). As a previous step to study
the molecular properties of the functionalized haptens, the lowest-
energy conformers of PY in water were determined using computer
assisted techniques. This molecular search resulted in a large
amount of conformers (see Figure 1SI†), which were processed
and analyzed as described in the experimental part (see that section
for determination of minimum-energy conformations). Fig. 3
depicts the optimized geometry (PM3-H2O) of the conformer
that displayed the lowest energy value (refer to Figure 2SI† for
the geometries of the other minimum-energy conformers, their
classification and selection). Interestingly, the PY molecule, like
the natural strobilurin A and several of its analogues,14 does not
adopt an extended geometry in solution. All minimum-energy
conformations were characterized by two planar moieties—one
plane was formed by the chlorophenyl and pyrazolyloxy moieties
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360◦ in 15◦ increments. From this study, we observed that the
minimum energy conformers for haptens PYo5 and PYs5 showed a
trend to display the linker away from the backbone of the molecule.
On the contrary, the lowest energy conformers of PYa6 showed
that the linker has a certain tendency to bend over the molecule
(Fig. 5), a finding that provides a feasible explanation for the
inferior performance of the PYa6 derivative as immunizing hapten.
Conclusions
In order to obtain antibodies endowed with appropriate binding
properties to small organic molecules, optimal functionalized
derivatives of the target analyte must be prepared. In the present
study, multistep synthetic routes were carried out for a better un-
derstanding of the importance of the derivatization site. Molecular
modeling studies indicated that, in the case of haptens PYo5 and
PYs5, the spacer arm facilitated an adequate exposure of the
skeleton of PY during the immune response. On the contrary,
the central position of the linker in the molecule of PYa6 could
have caused steric hindrance that impaired the formation of a
deep and tight binding pocket, typical of most antibody–hapten
interactions.17 Therefore, interactions between the linker arm and
the backbone of the molecule may occur not only if too long
spacers are employed, as demonstrated in our previous paper,5 but
also because of an inappropriate tethering site. Acquisition of a
larger body of knowledge and more profound studies are required
in order to better understand the factors influencing the formation
and stabilization of the antibody–analyte complex. In practice,
these antisera—particularly those derived from haptens PYo5 and
PYs5—should become very useful reagents for the preparation
of immunoaffinity columns intended for PY purification and
concentration, as well as for the development of immunochemical
methods enabling the selective and sensitive determination of PY
in incurred real samples.
Fig. 4 Computed partial atomic charges of PY and haptens PYo5, PYa6
and PYs5. Only those atoms showing a charge variation higher than 1%
between PY and any of the haptens are depicted. Hydrogens are not
included.
electronic similarities between PY and the immunizing haptens
were found. In hapten PYs5, the hydrocarbon spacer chain is teth-
ered to the benzene ring through a sulfur bridge that replaced the
chlorine atom of PY. This substitution meant the highest observed
modification of the electronic properties compared with the two
other haptens (the partial charge at C1 in PYs5 changed by 37%).
Nevertheless, this sort of replacement, which has been previously
used for the design of haptens for chlorine-containing analytes,16 is
less drastic than if a C–C bond had been formed. In addition, the
antibody with the highest affinity to PY was derived from hapten
PYs5, so electronic charges did not account by themselves for the
observed differences among immunizing haptens.
As a further step to understand the reason why hapten PYa6
was a poorer mimic of the PY molecule than haptens PYo5 and
PYs5, the structure of the three synthesized haptens was modeled
taking into account the flexibility of the spacer arm. In order to
simplify this analysis, we assumed that in the synthetic haptens the
common PY framework can adopt a conformational disposition
similar to that of PY itself. A preliminary estimation of the
preferred orientation of the spacer arm in each hapten was done
by calculating the conformer with the lowest energy in aqueous
solution when the two first C–C bonds of the spacer arm proximal
to the backbone of the PY molecule were rotated independently
Experimental
PY
[methyl
{2-[1-(4-chlorophenyl)pyrazol-3-yloxymethyl]-
phenyl}methoxy carbamate] (CAS Registry No. 175013-18-0,
Fig. 5 Most probable geometries and electron density surfaces colored by electrostatic potential from two different views of the three synthesized haptens.
Color codes are the same as in Fig. 3.
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MW 387.8 g mol-1) was purchased, Pestanal grade, from
Riedel-de-Hae¨n (Seelze, Germany). The methyl methoxy(o-
tolyl)carbamate (1) used in this work was kindly provided by
BASF AG (Limburgerhof, Germany). tert-Butyl hex-5-ynoate
(8) and tert-butyl 5-bromopentanoate (15) were prepared
from commercial hex-5-ynoic acid18 and 5-bromopentanoic
acid, respectively.19 Methyl 2-(bromomethyl)phenyl(methoxy)
carbamate (18) was prepared from tolylcarbamate 1.20 All melting
points were determined using a Kofler hot-stage apparatus or a
Bu¨chi melting point apparatus and are uncorrected. All NMR
spectra were recorded in CDCl3 or acetone-d6 at rt on a Bruker
method PM3 with COSMO water simulation using dihedral
angle increments of 15◦. Electrostatic potential energy surfaces
were mapped over electron density isosurfaces using the same
force field. The electron density probability value used for all
-3
˚
calculations was 0.01 electrons A .
Buffers and solutions
The following buffers were employed: PB, 100 mM sodium
phosphate buffer, pH 7.4; PBS, 10 mM sodium phosphate buffer,
pH 7.4 with 140 mM NaCl; PBST, PBS containing 0.05% (v/v)
Tween 20; CB, 50 mM carbonate–bicarbonate buffer, pH 9.6; and
washing solution, 150 mM NaCl containing 0.05% Tween 20. PY
and all other analytes were prepared as concentrated solutions
in N,N-dimethylformamide (DMF) and kept at -20 ◦C in amber
glass vials.
1
AC-300 spectrometer (300.13 MHz for H and 75.47 MHz for
13C). The spectra were referenced to residual solvent protons
in the 1H NMR spectra (7.26 and 2.05 ppm) and to solvent
carbons in the 13C NMR spectra (77.0 and 30.83 ppm). Infrared
spectra were measured as thin films between NaCl plates for
liquid compounds and as KBr pellets for solids using a Nicolet
Avatar 320 spectrometer. Electron-impact (EI) and fast atom
bombardment (FAB) mass spectra (MS and HRMS) were
obtained with a Micromass VG Autospec spectrometer. ELISA
absorbances were read with a PowerWave HT microplate reader
from BioTek Instruments (Winooski, VT, USA). A Voyager
De-Pro workstation from Applied Biosystems was used for
the analysis of the molecular weight of protein conjugates by
matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-TOF-MS).
Hapten synthesis
The synthesis of hapten PYo5 was previously published.7 Hapten
PYa6 was synthesized from methyl methoxy(o-tolyl)carbamate
(1)20 following the synthetic route detailed in Scheme 1 and
hapten PYs5 was synthesized from commercial methyl (4-
iodophenyl)hydrazine (12) following the procedure depicted in
Scheme 2. Unless otherwise stated, organic extracts were succes-
sively washed with H2O and brine, dried over anhydrous Na2SO4,
and concentrated under vacuum. The characterization data of the
synthetic intermediates can be found in the ESI.† All solvents were
purified by distillation and, if required, they were dried according
to standard methods.
Determination of minimum-energy conformations
The initial generation of low-energy conformations and geometry
optimizations of PY was made using the conformational space
search program CONFLEX6 from Conflex Corp. (Tokyo, Japan)
running on Windows XP. The visualization and analysis interface
BARISTA, a platform especially designed for conformational
analyses, was used to create the initial parameters, and con-
formation search was carried out with MMFF94S (2006-11-
24HGTEMP) parameters using the default configuration re-
sources, except for the upper limit value of conformational search
that was set to cover an area of 10 kcal mol-1 within the most
stable conformation (SEL = 10).21 The GBSA continuum solvation
method was also introduced to evaluate the solvation energy
contribution to the steric energy.22 The energy distribution of
conformers was established under steric energy analysis. Those
conformations that were chemically significant (accounting for
99.8% of the computed population) were grouped based on dihe-
dral angle values (refer to Table 1SI in the ESI†) and the lowest-
energy conformation of each group was used to create a potential
energy map in which the two dihedral angles differing within
each group were rotated independently 360◦ in 10◦ increments
using the PM3 semiempirical method, including the solvent effects
of water as simulated by the COSMO solvation model. The
potential energy conformation maps were constructed for each
selected conformation with CAChe WorkSystem Pro from Fujitsu
Ltd. (Tokyo, Japan) using the MOPAC_OpMap2_PM3_H2O
procedure. Several of the low-energy conformations obtained from
each generated potential energy map were chosen and reoptimized,
and the lowest-energy conformations thus obtained were selected
as the minimum energy conformations of PY. Optimized geome-
tries of haptens were also calculated using the semi-empirical
Synthesis of PYa6
Methyl 4-iodo-2-methylphenyl(methoxy)carbamate
(2).
Ag2SO4 (1.097 g, 3.51 mmol) and I2 (0.893 g, 3.52 mmol)
were successively added to a solution of methyl methoxy(o-
tolyl)carbamate (1) (680 mg, 3.48 mmol) in glacial acetic acid
(9 mL), and the reaction mixture was stirred for 20 h at rt in the
dark. After this time, a yellowish precipitate of AgI was formed,
which was removed by filtration. The filtrate and washes were
diluted with water and extracted with CH2Cl2. The combined
organic extracts were successively washed with aqueous NaHCO3,
aqueous Na2S2O7, brine, and dried. Evaporation of the solvent
under vacuum gave 2 (1.093, 98%) as a viscous oil that had a
1
purity greater than 95%, as judged by H NMR analysis, and it
was used in the next step without further purification.
Methyl 2-(bromomethyl)-4-iodophenyl(methoxy)carbamate (3).
A solution of methoxycarbamate 2 (810 mg, 2.50 mmol) and
bromine (3.21 g, 1.03 mL, 20.1 mmol, 8 equiv) in dry CCl4
(90 mL) was irradiated with a mercury lamp (125 W) for 36 h
at rt. The reaction mixture was washed with aqueous Na2S2O7
and brine, and dried. The solvent was evaporated under vacuum
to give an oily residue (885 mg) consisting in a mixture of
monobrominated compound 3, the corresponding dibrominated
derivative, and unreacted starting material that could not be
separated by conventional chromatographic purification methods.
The percent of 3 in the mixture was estimated to be 50–55% on
the basis of the analysis of the mixture by NMR.
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1-(4-Chlorophenyl)-1H-pyrazol-3-ol (6). A solution of sodium
ethoxide in ethanol, prepared by dissolving Na (882 mg, 38.3 mol)
in anhydrous ethanol (27 mL), was added to a suspension of 4-
chlorophenylhydrazine hydrochloride (4) (3.00 g, 16.75 mmol) in
dry toluene (27 mL) stirred under nitrogen at 40 ◦C. After stirring
at the same temperature for 10 min, methyl acrylate (7.21 g,
7.54 mL, 83.75 mmol) was added drop wise during 20 min and
the resulting mixture was stirred for 1.5 h. The reaction mixture
was concentrated in a rotary evaporator to remove most of the
ethanol, then diluted with water (300 mL) and extracted with
benzene. The benzene extracts were extracted with 5% sodium
hydroxide and the combined aqueous basic phases were acidified
with acetic acid to pH 6 and extracted with benzene. The organic
layer was washed, dried, and concentrated to give nearly pure 1-(4-
chlorophenyl)pyrazolidin-3-one (5) (2.35 g, 71%) as an amorphous
solid.
reaction mixture was stirred at rt for 16 h. The solvent was removed
under vacuum, and the residue was purified by chromatography,
using hexane–ethyl acetate 9 : 1 as eluent, to furnish compound 10
(220 mg, 92%).
6-(3-((1-(4-Chlorophenyl)-1H-pyrazol-3-yloxy)methyl)-4-
(methoxy(methoxycarbonyl)amino)phenyl)hexanoic acid (11, hap-
ten PYa6). A solution of the tert-butyl ester 10 (203 mg,
0.364 mmol) in trifluoroacetic acid (1.5 mL) and CH2Cl2 (1.5 mL)
was stirred at 0 ◦C until TLC [developed with hexane–ethyl acetate
(7 : 3)] showed completion of the reaction (about 1 to 1.5 h). The
reaction mixture was concentrated under vacuum without heating
and the residue obtained was purified by silica gel chromatography,
using 4% of methanol in CHCl3 as eluent, to give the hapten PYa6
(11) (182 mg, 99%) as a slightly colored viscous oil; dH (CDCl3)
7.70 (1H, d, J = 2.6 Hz, H-5 Pz), 7.55 (2H, m, part AA¢ of the
AA¢BB¢ system, J = 8.8, 2.5, 0.5 Hz, H-2, and H-6 ClPh), 7.47
(1H, d, J = 1.8 Hz, H-3 Ph), 7.37 (2H, m, part BB¢ of the AA¢BB¢
system, J = 8.8, 2.5, 0.5 Hz, H-3, and H-5 ClPh), 7.27 (1H, d,
J = 8.1 Hz, H-6 Ph), 7.18 (1H, dd, J = 8.1, 1.8 Hz, H-5 Ph), 5.94
(1H, d, J = 2.6 Hz, H-4 Pz), 5.30 (2H, s, OCH2), 3.79 (3H, s,
CO2CH3), 3.75 (3H, s, NOCH3), 2.66 (2H, t, J = 7.7 Hz, H-6),
2.34 (2H, t, J = 7.4 Hz, H-2), 1.64 (4H, m, H-3 and H-5) and 1.40
(2H, m, H-4); dC (CDCl3) 179.01 (C-1), 164.30 (C-3 Pz), 155.94
(NCO2), 143.46 (C-1 Ph), 138.59 (C-1 ClPh), 134.92 and 134.40
(C-2 and C-4 Ph), 130.61 (C-4 ClPh), 129.34 (C-3 and C-5 ClPh),
128.87 and 128.58 (C-3 and C-5 Ph), 127.73 (C-5 Pz), 127.17 (C-6
Ph), 118.93 (C-2 and C-6 ClPh), 94.33 (C-4 Pz),66.95 (OCH2),
61.94 (NOCH3), 53.52 (CO2CH3), 35.28 (C-6), 33.80 (C-2), 30.69
(C-5), 28.47 (C-4) and 24.43 (C-3); umax/cm-1 (NaCl) 3400–2800,
2933, 2861, 1735, 1707, 1546, 1501, 1481, 1358, 1094 and 751;
m/z (EI) 441 (8%), 440 (6), 439 (22), 308 (9), 278 (10), 277 (18),
247 (16), 277 (17), 276 (10), 246 (100) and 194 (59); m/z (FAB)
calcd for C25H29ClN3O6 (M+ + H) 502.17449, found 502.17539;
lmax (PB)/nm 280 (e/dm3 mol-1 cm-1 12.1) and 260 (11.7).
The above solid (1.265 g, 6.43 mmol) and CuCl (68 mg,
0.32 mmol) were dissolved in DMF (13 mL) and O2 was bubbled
through the mixture during 3 h at rt. The reaction mixture was
diluted with H2O (100 mL) and then stirred for 1 h. The formed
precipitate was filtered off, washed with water, and dried to afford
a brownish solid that was crystallized from benzene to give 6
◦
(993 mg, 80%) as a slightly colored solid; mp 190–192 C (from
benzene), at 174–176 ◦C flake-like crystals were transformed into
needle like crystals (lit.,10 mp 181–182 ◦C).
Methyl 2-((1-(4-chlorophenyl)-1H-pyrazol-3-yloxy) methyl)-4-
iodophenyl(methoxy)carbamate (7). A mixture of pyrazolol 6
(186.3 mg, 0.96 mmol), benzyl bromide 3 (768 mg of the mixture
obtained above from the bromination of methoxycarbamate 2,
containing approximately 1.0 mmol of 3), and CsCO3 (759 mg,
2.32 mmol) in anhydrous DMF (6 mL) was stirred at rt overnight.
The reaction mixture was diluted with H2O and extracted with
ethyl acetate. The organic layer was washed and dried. Filtration
and evaporation of the solvent was followed by column chro-
matography, using hexane–ethyl acetate 8 : 2 as eluent, to give 7
(395 mg, 80%) as an oil that solidified upon standing; mp 105–
107 ◦C (from methanol).
Synthesis of PYs5
1-(4-Iodophenyl)-1H-pyrazol-3-ol (14). A 1 M solution of
potassium tert-butoxide in tert-butanol (1.2 mL, 1.2 mmol) was
added to a solution of (4-iodophenyl)hydrazine (12) (234 mg,
1 mmol) in anhydrous toluene (1.2 mL) at rt under nitrogen. The
mixture was warmed to 50 ◦C and then drop wise treated with tert-
butyl acrylate (135 mg, 153 mL, 1.05 mmol). The reaction mixture
tert-Butyl 6-(3-((1-(4-chlorophenyl)-1H-pyrazol-3-yloxy) me-
thyl)-4-(methoxy(methoxycarbonyl)amino)phenyl)hex-5-ynoate
(9). Dry DMF (1.5 mL) and triethylamine (1.2 mL) were added
to a mixture of aryl iodide 7 (241.5 mg, 0.468 mmol), tert-butyl
hex-5-ynoate (8) (117.9 mg, 0.702 mmol), Cl2Pd(PPh3)2 (9.3 mg,
0.13 mmol), and CuI (3.6 mg, 0.19 mmol) under nitrogen. The
mixture was degassed through several freeze–thaw cycles, stirred
at rt for 30 min and then at 60 ◦C for 4 h. The resulting
brownish reaction mixture was cooled down, filtered through
cotton and most of the triethylamine was removed under vacuum.
The residue was diluted with ethyl acetate and washed, dried,
and concentrated. Purification by silica gel chromatography, using
hexane–ethyl acetate 9 : 1 as eluent, afforded compound 9 (255 mg,
98%) as an oil.
◦
was stirred at 50 C for a few minutes, then cooled to rt, diluted
with benzene, and extracted with 5% aqueous KOH. The aqueous
layer was treated with excess CO2 and the fuchsia solid formed was
extracted with ethyl acetate (5 times). The combined organic layers
were washed and dried. Filtration and evaporation of the solvent
afforded a solid that was crystallized from benzene–acetone to
give crystals of 1-(4-iodophenyl)pyrazolidin-3-one (13) (179 mg,
62%) as a brown solid; mp 142–143 ◦C (from benzene–acetone).
A mixture of the compound 13 obtained above (164 mg,
0.577 mmol) and CuCl (2.5 mg, 0.03 mmol) in DMF (2.5 mL)
was stirred in the air until all the starting material was consumed
(about 5 h). The reaction mixture was diluted with H2O and the
solid formed was collected by filtration, washed with cold water,
and dried under vacuum to obtain pure 1-(4-iodophenyl)-1H-
pyrazol-3-ol (14) (160 mg, 97%) as a cream color solid; needle-like
tert-Butyl 6-(3-((1-(4-chlorophenyl)-1H-pyrazol-3-yloxy) me-
thyl)-4-(methoxy(methoxycarbonyl)amino)phenyl)hexanoate (10).
A solution of alkyne 9 (243 mg, 0.439 mmol) and Wilkinson’s
catalyst (12.2 mg, 0.013 mmol, 3%) in tetrahydrofuran (3 mL) was
evacuated and purged under an atmosphere of hydrogen gas. Then,
the hydrogen pressure was regulated to 4 atmospheres and the
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◦
crystals from hexane–ethyl acetate began to sublimate at 190 C
for C24H27N3O6S 485.16206, found 485.16249; lmax (PB)/nm 280
(e/dm3 mol-1 cm-1 19.2) and 260 (8.15).
and melted at 222–224 ◦C.
tert-Butyl
5-(4-(3-hydroxy-1H-pyrazol-1-yl)phenylthio)pen-
Protein conjugate preparation
tanoate (17). tert-Butyl 5-mercaptopentanoate (16) was
prepared from tert-butyl 5-bromopentanoate (15) and thiourea
with very high yield via formation of the thiouronium salt
followed by cleavage with sodium pyrosulfite.11 Then, a mixture of
iodophenyl-pyrazolol 14 (200 mg, 0.70 mmol), thiol 16 (200 mg,
1.05 mmol), K2CO3 (193 mg, 1.40 mmol), CuI (7 mg, 0.035 mmol),
and ethylene glycol (78 mL, 1.40 mmol) in 2-propanol (0.7 mL)
was degassed through three freeze–pump–thaw cycles, sealed
under vacuum, and heated at 80–85 ◦C with stirring for 24 h.
The cooled reaction mixture was diluted with ethyl acetate and
washed, dried, and concentrated. The brownish oily residue was
purified by column chromatography, using hexane–ethyl acetate
mixtures from 9 : 1 to 7 : 3 as eluent, to give, in order of elution,
unreacted starting pyrazolol 14 (54 mg, 73% conversion) and
compound 17 (130 mg, 70%) as a solid; mp 113–114 ◦C (from
hexane–isopropanol).
All of the synthesized haptens contained a free carboxylic group
for protein conjugation. The hapten-to-protein MR of each bio-
conjugate was calculated by MALDI-TOF-MS and by differential
absorbance measurements. For MALDI-TOF-MS analysis, a frac-
tion of each conjugate solution was extensively dialyzed against
pure water at 4 ◦C and lyophilized. Samples were resuspended in
acetonitrile–water 7 : 3 containing 0.1% trifluoroacetic acid, and
they were charged into the plate in between two layers of sinapinic
acid.
Immunizing conjugates
BSA conjugates were prepared by the active ester proce-
dure. Briefly, 20 mmol of hapten in DMF was mixed with
20 mmol of N-hydroxysuccinimide and 20 mmol of N,N-
dicyclohexylcarbodiimide also in DMF. Additional DMF was
added to bring the final concentration of all reagents to
50 mM. Hapten activation was left to occur overnight at rt in
amber vials. The day after, the reaction was centrifuged and
the supernatant was collected. Next, 200 mL of activated hapten
solution was added drop wise to 1.0 mL of a 15.0 mg mL-1 BSA
solution in CB. The coupling reaction was allowed to happen
during 4 h at rt with moderate stirring. The initial hapten-to-
protein MR in the reaction mixture was approximately 44 : 1.
Finally, the conjugate was separated from uncoupled hapten by
gel filtration on Sephadex G-25, using PB as eluent. The purified
bioconjugate was diluted to 1.0 mg mL-1 with PB and stored at
-20 ◦C. For differential absorbance measurements, the absorbance
values of the conjugate at 280 and 260 nm were considered.
tert-Butyl 5-(4-(3-(2-(methoxy(methoxycarbonyl)amino) benzyl-
oxy)-1H-pyrazol-1-yl)phenylthio)pentanoate (19). A mixture of
compound 17 (99 mg, 0.284 mmol), methyl 2-(bromomethyl)-
phenyl(methoxy)carbamate (18, prepared bybenzylicbromination
of tolylcarbamate 1 with N-bromosuccinimide and azaisobuty-
ronitrile in CCl4 at reflux)8 (117 mg, 0.427 mmol), and CsCO3
(139 mg, 0.427 mmol) in anhydrous DMF (1.2 mL) was stirred at
rt overnight under nitrogen. The reaction mixture was diluted with
ethyl acetate and washed, dried, filtered, and then evaporated to
dryness. Purification by column chromatography, using hexane–
ethyl acetate 9 : 1 as eluent, afforded compound 19 (134 mg, 89%)
as a viscous oil.
5-(4-(3-(2-(Methoxy(methoxycarbonyl)amino)benzyloxy)-1H-
pyrazol-1-yl)phenylthio)pentanoic acid (20, hapten PYs5). A so-
lution of the tert-butyl ester 19 (107 mg, 0.197 mmol) in a 1 : 1
mixture of trifluoroacetic acid and CH2Cl2 (4 mL) was stirred at
0 ◦C for 1 h. Work-up of the reaction mixture as described above
for the hydrolysis of 10 to 11, followed by chromatography on silica
gel and using CHCl3–methanol 95 : 5 as eluent, afforded hapten
PYs5 (20) (86 mg, 90%) as a colorless viscous oil; dH (CDCl3) 7.69
(1H, d, J = 2.6 Hz, H-5 Pz), 7.66 (1H, m, H-4 Ph), 7.51 (2H, m,
part AA¢ of the AA¢BB¢ system, J = 8.8, 2.5, 0.5 Hz, H-2 and H-6
SPh), 7.40–7.35 (3H, m, H-3, H-5 and H-6 Ph overlapped with
the part BB¢ of the AA¢BB¢ system corresponding to H-3 and H-5
SPh), 5.90 (1H, d, J = 2.6 Hz, H-4 Pz), 5.34 (2H, s, OCH2), 3.79
(3H, s, CO2CH3), 3.75 (3H, s, NOCH3), 2.91 (2H, t, J = 7.1 Hz,
H-5), 2.36 (2H, t, J = 7.0 Hz, H-2) and 1.72 (4H, m, H-3 and
H-4); dC (CDCl3) 178.86 (C-1), 164.10 (C-3 Pz), 155.84 (NCO2),
138.48 and 137.30 (C-1 and C-2 Ph), 134.77 (C-1 SPh), 132.69
(C-4 SPh), 130.95 (C-3 and C-5 SPh), 128.85, 128.80 and 128.48
(C-3, C-4 and C-5 Ph), 127.66 (C-5 Pz), 127.01 (C-6 Ph), 118.29
(C-2 and C-6 SPh), 94.05 (C-4 Pz), 66.79 (OCH2), 62.09 (NOCH3),
53.54 (CO2CH3), 33.99 (C-5), 33.35 (C-2), 28.33 (C-4) and 23.58
(C-3); umax/cm-1 (NaCl) 3500–2800, 3142, 3044, 2954, 1793, 1708,
1545, 1480, 1358, 1267, 1100 and 737; m/z (EI) 485 (M+, 0.1%),
467 (1), 454 (1.5), 453 (4), 437 (6), 425 (7), 424 (18), 423 (76),
422 (4), 379 (3), 291 (30), 162 (12) and 132 (100); m/z (EI) calcd
Coating conjugates
In this case, the coupling reaction of the hapten to the carrier
protein was accomplished by the mixed anhydride procedure.
Basically, 18 mmol of hapten was dissolved in 180 mL of DMF
and mixed with 18 mmol of tributylamine and 18 mmol of isobutyl
chloroformate also in DMF. The concentration of all reagents
was brought to 90 mM with DMF and the hapten was left to
be activated during 1 h at rt. Next, 100 mL of activated hapten
solution was added drop wise to 2.0 mL of a 15.0 mg mL-1 OVA
solution in CB. The coupling reaction was carried out during 2.5 h
at rt with moderate stirring. The initial hapten-to-protein MR in
the reaction mixture was approximately 13 : 1. Conjugates were
processed and stored as described above.
Tracer conjugates
The mixed anhydride method was also used to prepare the
corresponding enzyme conjugates. First, haptens were activated
as described above and a 1/10 dilution in DMF was prepared
after the incubation step. Next, 100 mL of this dilution was
added drop wise to a 1.0 mL solution of HRP at 2.2 mg
mL-1 in CB. The coupling reaction was incubated for 4 h at
rt with moderate stirring. The initial hapten-to-protein MR in
the reaction mixture was approximately 18 : 1. The tracer was
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separated from uncoupled hapten by gel filtration on Sephadex
G-25. For differential absorbance measurements, the absorbance
values of the conjugate at 400 and 280 nm were considered in this
case. The purified tracer was brought to 1.0 mg mL-1 with PB and
diluted 1/2 with PBS containing 1% (w/v) BSA and 0.01% (w/v)
thimerosal. The conjugate was stored at -20 ◦C in amber glass
vials for long-term preservation and a working aliquot was kept
at 4 ◦C.
Antibody-coated direct assays
Microplates were coated with 100 mL per well of a 1/3000,
1/10000, and 1/30000 dilution of a given antiserum in CB by
overnight incubation at rt. After four washes, 50 mL per well
of PY standard curve was dispensed to each plate column and
immediately 50 mL per well of a different concentration of a
given enzyme tracer was added. In this case, serial three-fold
dilutions in PBST of the HRP conjugate (from 600 to 2 ng mL-1)
were prepared. The same array of reagent concentrations was
distributed in another plate that had previously been coated with a
different antiserum. The immunological reaction was carried out
during 1 h at rt and the enzyme activity was revealed as described
before.
Antiserum production
Animal manipulation was performed in compliance with the
laws and guidelines of the Spanish Ministry of Science and
Innovation (RD 1201/2005 and law 32/2007) and according to
the European Directive 2003/65/EC (amending general Council
Directive 86/609/EEC) concerning the protection of animals
used for experimental and other scientific purposes. Two New
Zealand white female rabbits weighing 1–2 kg were immunized
by subcutaneous injection with 0.3 mg of conjugate BSA–PYo5,
BSA–PYa6, or BSA–PYs5 following described procedures.23 For
further details see the ESI.†
Acknowledgements
We thank Joaquin J. Barjau for the preparation of some of the in-
termediates used in the synthesis of the haptens, and Laura Lo´pez-
Sa´nchez and Ana Izquierdo-Gil for excellent technical assistance.
We also thank Dr Reinhard Kirstgen from BASF for kindly
providing strobilurin analogues and hapten synthesis precursors.
This work was supported by Ministerio de Educacio´n y Ciencia
(AGL2006-12750-C02-01/02/ALI) and cofinanced by FEDER
funds. J.V.M. was hired by the CSIC under a Ramo´n y Cajal
contract financed by Ministerio de Ciencia e Innovacio´n and the
European Social Fund.
Competitive immunoassays
All antisera were evaluated in the two classical cELISA formats
using both homologous and heterologous conjugates. Eight-
channel electronic pipettes were employed for rapid and precise
liquid dispensing. Microplates were washed four times with
washing solution using a 96-channel ELx405 washer from BioTek
Instruments (Winooski, VT, USA). After the assay, the absorbance
was read at 492 nm with a reference wavelength at 650 nm.
Sigmoidal curves were mathematically fitted to a four-parameter
logistic equation using the SigmaPlot software package from SPSS
Inc. (Chicago, IL, USA). The antiserum titer was defined as the
reciprocal of the dilution that results in a maximum absorbance
value (Amax) around 1.0 reached at the zero dose of analyte when
assayed using the indirect competitive format with the homologous
conjugate at 1.0 mg mL-1. Antibody affinity was estimated as the
concentration of analyte that reduced 50% (IC50) the Amax value,
and the assay limit of detection was defined as the IC10 of the
inhibition curve.
Notes and references
1 K. Landsteiner, Koninkl. Akad. Wet. Amst. Verslag, 1921, 30,
329.
2 For example, (a) S. Lu, Y. Zhang, J. Liu, C. Zhao, W. Liu and R. Xi,
J. Agric. Food Chem., 2006, 54, 6995; (b) J. V. Mercader, C. Sua´rez-
Pantaleo´n, C. Agullo´, A. Abad-Somovilla and A. Abad-Fuentes,
J. Agric. Food Chem., 2008, 56, 2581.
3 (a) Y. J. Kim, Y. A. Cho, H. S. Lee, Y. T. Lee, S. J. Gee and B. D.
Hammock, Anal. Chim. Acta, 2003, 475, 85; (b) Y. H. Liu, M. J. Jin,
W. J. Gui, J. L. Cheng, Y. R. Guo and G. N. Zhu, Anal. Chim. Acta,
2007, 591, 173.
4 (a) Z. Wang, Y. Zhu, S. Ding, F. He, R. C. Beier, J. Li, H. Jiang, C.
Feng, Y. Wan, S. Zhang, Z. Kai, X. Yang and J. Shen, Anal. Chem.,
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