Structure-immunogenicity relationship of kresoxim-methyl regioisomeric haptens

Article abstract of DOI:10.1039/c3ob41570h

Kresoxim-methyl was one of the two first strobilurins to be discovered, and nowadays it is widely used as an antifungal agent in crop protection. Because of its low molecular weight and negligible structural complexity, the generation of antibodies to kresoxim-methyl noticeably requires the preparation of functionalized haptens. In this study, the introduction of a hydrocarbon spacer arm at the aromatic moieties of the target molecule was carried out by a convergent strategy based on the Sonogashira cross-coupling reaction, and functionalized linkers of the same length were also tethered to the aliphatic toxophore group by the O-alkylation reaction. Evaluation of the immune response, in terms of antibody affinity, showed a differential behavior among five synthesized haptens whose sole dissimilarity was the derivatization site. The characteristic (methoxyimino)acetate moiety of strobilurins was revealed as the optimum linker position for high-affinity polyclonal and monoclonal antibody production. However, good monoclonal antibodies were isolated from mice immunized with a hapten carrying the linker at an opposite site, which otherwise generated a poor polyclonal response in rabbits. Site-heterology was confirmed as a feasible approach for the improvement of the apparent affinity, particularly with polyclonal antibodies. Several of the monoclonal antibodies generated in the context of this project could be proper binders for kresoxim-methyl immunosensing over different analytical platforms.

Full text of DOI:10.1039/c3ob41570h

Organic &
Biomolecular Chemistry
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Structure–immunogenicity relationship of
kresoxim-methyl regioisomeric haptens†
Cite this: Org. Biomol. Chem., 2013, 11,
361
7
a
b
a
Rosario López-Moreno,‡ Josep V. Mercader,‡ Consuelo Agulló,
a
b
Antonio Abad-Somovilla* and Antonio Abad-Fuentes*
Kresoxim-methyl was one of the two first strobilurins to be discovered, and nowadays it is widely used as
an antifungal agent in crop protection. Because of its low molecular weight and negligible structural
complexity, the generation of antibodies to kresoxim-methyl noticeably requires the preparation of func-
tionalized haptens. In this study, the introduction of a hydrocarbon spacer arm at the aromatic moieties
of the target molecule was carried out by a convergent strategy based on the Sonogashira cross-coupling
reaction, and functionalized linkers of the same length were also tethered to the aliphatic toxophore
group by the O-alkylation reaction. Evaluation of the immune response, in terms of antibody affinity,
showed a differential behavior among five synthesized haptens whose sole dissimilarity was the derivati-
zation site. The characteristic (methoxyimino)acetate moiety of strobilurins was revealed as the optimum
linker position for high-affinity polyclonal and monoclonal antibody production. However, good mono-
clonal antibodies were isolated from mice immunized with a hapten carrying the linker at an opposite
site, which otherwise generated a poor polyclonal response in rabbits. Site-heterology was confirmed as
a feasible approach for the improvement of the apparent affinity, particularly with polyclonal antibodies.
Several of the monoclonal antibodies generated in the context of this project could be proper binders
for kresoxim-methyl immunosensing over different analytical platforms.
Received 31st July 2013,
Accepted 6th September 2013
DOI: 10.1039/c3ob41570h
www.rsc.org/obc
to the molecule and holds both the toxophore moiety and a
distinctive bulky substituent in the ortho position. Kresoxim-
Introduction
3
Strobilurins are one of the most important families of modern methyl, which was released in the mid 1990s by BASF, belongs
crop protection compounds. Their invention was made pos- to the first generation of strobilurins. Structurally, kresoxim-
sible after the discovery and chemical description, by Anke methyl is characterized by a methyl (E)-2-(methoxyimino)-
1
and coworkers in 1977, of strobilurin A – a natural antibiotic 2-phenylacetate group (a-ring), which holds the toxophore
derivative of β-methoxyacrylic acid that was isolated from moiety responsible for its biocide activity and an o-tolyl group
Strobilurus tenacellus and Oudemansiella mucida. More recently, (b-ring) – both rings being joined together by means of a
it was discovered that strobilurins are also produced by other methylidenoxy group (Fig. 1). At 20 °C, kresoxim-methyl is a
−1
genera, such as Xerula, Hydropus, Mycena, Filoboletus, Crepido- white solid with low solubility in water (2 mg L ) and low
2
−8
tus, Cyphellopsis, and Bolinea. However, the naturally secreted volatility (vapor pressure of 1.72 × 10 mmHg), and it is
product is not stable to light and therefore not useful for pest highly photostable, showing a half-life time of 30 days. The
control. All currently registered strobilurins – 19 so far – are synthetic pathway to prepare this molecule evolved from the
4
synthetic nature-derived chemicals, which comprise
a
first procedure described by Wenderoth et al. in 1989, which
common aromatic bridge that provides photochemical stability implied several synthetic steps for the construction of the
methoxyimino)acetate moiety, to more recent strategies based
(
on the Suzuki–Miyaura coupling reaction that allow the stereo-
selective construction of the (E)-2-(methoxyimino)-2-phenyl-
acetate moiety with high yield in only one synthetic step.
Antibody-based techniques nowadays constitute valuable
alternative strategies for small organic chemical monitoring.
In particular, antibodies recognizing modern and relevant pes-
ticides are deemed valuable tools for the development of rapid
analytical methods enabling a more efficient control of chemi-
cal residues in foodstuffs and the environment. Since low
a
Department of Organic Chemistry, Universitat de València, Doctor Moliner 50,
6100 Burjassot, València, Spain. E-mail: antonio.abad@uv.es; Fax: +34-963544328;
4
5
Tel: +34-963544509
b
Institute of Agrochemistry and Food Technology, Consejo Superior de Investigaciones
Científicas (IATA–CSIC), Agustí Escardino 7, 46980 Paterna, València, Spain.
E-mail: aabad@iata.csic.es; Fax: +34-963636301; Tel: +34-963900022
†
Electronic supplementary information (ESI) available: Full characterization
data of all the intermediate compounds in the synthesis of haptens and copies
1
of H NMR spectra. See DOI: 10.1039/c3ob41570h
‡
These authors contributed equally to this work.
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Fig. 1 (A) Global minimum energy conformation of kresoxim-methyl. Calculations were performed using Molecular Mechanics (MM3) as implemented in the
CAChe program. A systematic conformational search was performed (all rotatable bonds were rotated by 15 degree steps) and the geometry of the generated con-
formers was refined by performing an optimized geometry calculation in MOPAC using PM3 parameters. Arrows denote the spacer-arm attachment site in each of
the haptens. (B) Structure of kresoxim-methyl (KM), the synthesized haptens, and the corresponding N-succinimidyl esters (activated haptens).
molecular weight compounds like kresoxim-methyl are not of the antibody binding site, and consequently influencing
2
7–34
able to trigger an immune response by themselves (so-called detectability and selectivity.
haptens), immunochemical methods rely on the availability of Functionalized haptens for kresoxim-methyl were previously
appropriate bioconjugates. Prior to antibody production, a prepared by our group through derivatization at the carboxyl
rational design of functionalized haptens for covalent coupling group of the (methoxyimino)acetate moiety, using as linkers
12
to carrier proteins has long been recognized as a critical step dipeptides or linear aliphatic chains of different lengths. In
for high-quality antibody generation and sensitive assay the present study, a thorough approach was undertaken in
6
–10
development.
Hapten functionalization aims at synthesiz- order to investigate the effect of the derivatization site on the
ing an analogue of the target compound which holds a spacer generation of high-quality antibodies for this chemical. Five
arm with a reactive or activatable chemical group at the free regioisomeric haptens – carrying identical spacers – were pre-
end of the linker. Thus, several criteria need to be considered pared with alternative rationally-chosen linker tethering sites,
before haptens are prepared, such as the functional moiety, thus covering a wide variety of spatial orientations of the target
the length and composition of the linker, and the derivatiza- molecule. A comprehensive study was undertaken so as to
tion site that will be most adequate for optimum immune clarify the influence of hapten orientation over the immune
response. For the functional group, carboxylates are mostly system response. Rabbit polyclonal antibodies (pAb) and mouse
preferred because they can easily be protected and deprotected monoclonal antibodies (mAb) were generated in order to evalu-
for chemical synthesis, and they can be readily activated for ate the antibody-eliciting capability of the different haptens.
high-yield covalent coupling to the abundantly available Antibody apparent affinity was first estimated using homolo-
ε-amine groups of lysine residues in proteins. The importance gous enzyme-linked immunosorbent assay (ELISA), and finally
of linker composition and length has been investigated by our the influence of tethering-site heterology was assessed for each
1
1–19
group and others.
Generally, linear aliphatic hydrocarbon antibody–hapten combination.
chains are preferred due to their low immunogenicity, and
medium-size spacers (four to six-carbon long) are mostly rec-
ommended for the generation of high-affinity and selective
Results and discussion
Hapten syntheses
2
0,21
antibodies,
although other linker lengths and compo-
2
2–26
sitions have also been employed.
Last but not least, the
linker tethering site decides the ultimate orientation of the The kresoxim-methyl framework exhibited significant confor-
hapten, thus determining upon immunization the formation mational flexibility due to the large number of bonds
7
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displaying free rotation. Calculations showed that, like natural C-alkylation reaction of the benzylic position with o-cresol fol-
3
5
strobilurins and several of its synthetic analogues, kresoxim- lowed by formic acid promoted hydrolysis of the tert-butyl
methyl does not adopt an extended geometry, so all minimum- ester group. The overall yield of the synthesis of hapten KMo
energy conformations are characterized by the nearly orthog- from starting material 18 was about 68%.
onal disposition of the two planar phenyl moieties (see Fig. 1A
for the most stable conformation). Particularly relevant is the
high degree of conformational flexibility of the (methoxy-
imino)acetate moiety, which can adopt several energetically
equivalent orientations by rotation around the C–C bond that
connects it to the aromatic backbone. On the basis of the
above conformational analysis, five different positions of the
kresoxim-methyl framework were selected for incorporation of
the linker, trying to cover as many spatial orientations of the
target molecule as possible (Fig. 1A and 1B). All of the
designed hapten structures maintained the complete hydro-
carbon skeleton and functional groups of the target analyte,
incorporating the same aliphatic spacer arm at positions
where minimum modifications of electronic, steric, and con-
formational features of the molecule were expected.
Protein conjugate preparation
Bioconjugates to BSA, OVA, and HRP were prepared with each
synthetic hapten using N,N′-disuccinimidyl carbonate as an
activating reagent. With this strategy, the active ester (NHS
ester, see Fig. 1) of the hapten could be easily purified. Since
pure activated haptens were employed for coupling, no
unwanted secondary reactions occurred during conjugation;
thus, the same chemical strategy could be followed both for
immunogen and for assay antigen preparation. Immunizing
conjugates showed hapten-to-protein molar ratios between 13
and 16, whereas molar ratios for OVA and HRP conjugates
were between 3 and 5; meaning that around half of the avail-
able lysine ε-amine groups of BSA, one-fourth of OVA, and all
those of HRP most likely held hapten molecules.
The synthesis of the haptens started from kresoxim-methyl
itself (haptens KMb and KMe) or from an aryl system that
already incorporated the (E)-2-(methoxyimino)acetate moiety
Immune response and polyclonal antibody characterization
(haptens KMa, KMc, and KMo). In all cases, the alkyl C-6 car- Two pAbs were generated from each immunogen, which were
boxylated spacer arm was introduced in the form of tert-butyl named after the immunizing hapten, and numbered. Every
ester, either through a Sonogashira cross-coupling reaction immunogen (BSA conjugates) was able to elicit a satisfactory
with the appropriate alkyne or by means of an O-alkylation immune response, with titers higher than 1/10 000 as deter-
reaction with the corresponding tert-butyl alkyl-bromoester. mined by direct ELISA. Evaluation of the affinity of antibodies
For all of the haptens, the synthesis was completed by acid raised by the different haptens was done by comparison of the
hydrolysis of the tert-butyl ester moiety.
IC50 values from assays with homologous conjugates, i.e.,
As shown in Fig. 2, the syntheses of haptens KMa, KMb, those carrying the same hapten as the immunogen but
and KMc followed a common strategy based on the initial coupled to HRP. The affinities of each pair of antisera that was
preparation of an iodinated derivative of kresoxim-methyl. The generated from the same immunizing hapten were essentially
rest of the steps to complete the synthesis of these haptens equivalent (Table 1), perhaps with the only exception of the
from the key iodinated intermediate (3, 7, or 12) implied palla- two pAbs produced from hapten KMa (rKMa#1 and rKMa#2),
dium/copper catalyzed Sonogashira cross-coupling reaction whose IC50 values differed 4-fold. The highest affinity to
with the terminal triple bond of tert-butyl hex-5-ynoate (4) (to kresoxim-methyl was found with KMo-type antibodies
give aryl-alkynes 5, 8, or 13), followed by hydrogenation of the (obtained from hapten KMo), followed by those triggered by
triple bond under homogeneous conditions using Wilkinson’s hapten KMe. Contrarily, antibodies with higher IC50 values
catalyst (to afford 6, 9, or 14), and finally, acidic hydrolysis of were produced from haptens containing the spacer arm at the
the tert-butyl ester group to provide haptens KMa, KMb, and b-ring (haptens KMb and KMc) or at the a-ring (hapten KMa).
KMc in 61%, 73%, and 55% overall yield, respectively, from Among these three haptens, probably the most surprising and
the corresponding starting materials.
unexpected result was the poor performance of KMb-type anti-
The synthesis of hapten KMe was readily accomplished in bodies because the position of the linker in hapten KMb
three steps (Fig. 3, left-hand side); i.e., alkaline hydrolysis of should not theoretically cause steric hindrance, and minimum
the methyl ester group of kresoxim-methyl to give carboxylic electronic or conformational modifications were involved.
acid 15, followed by O-alkylation of the carboxylate group with
The differences in kresoxim-methyl recognition exhibited
tert-butyl 6-bromohexanoate (16), and acid hydrolysis of the by the produced pAbs as a function of the hapten derivatiza-
tert-butyl ester group. This straightforward sequence afforded tion site can be observed in Fig. 4. In previous studies with
hapten KMe in nearly 60% overall yield from the starting other strobilurins, functionalization through the toxophore
material.
moiety, particularly at the methoxy group, also resulted in
3
6–38
Finally, for the synthesis of hapten KMo we made use of optimum affinity responses.
With the linker at such a
benzyl-bromide 19 (Fig. 3, right-hand side), prepared in high position, the characteristic aromatic rings of the molecule
yield from oxime 18 in two successive steps, first O-alkylation seem to be better exposed to the immune system. In contrast,
of the oxime moiety with tert-butyl 6-bromohexanoate (16) fol- the a-ring was revealed as a less adequate site for derivatiza-
lowed by radical bromination of the benzylic position with tion – particularly in the para position from the (methoxy-
NBS. The synthesis of hapten KMo was completed from 19 via imino)acetate group – if high-affinity antibodies are sought.
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Fig. 2 Synthetic sequence for the preparation of haptens KMa, KMb, and KMc.
It is well-known, particularly with pAbs, that changes in taking into account that linkers had been introduced at very
hapten structure can decrease the recognition of the corres- different positions of the molecular framework in the immu-
ponding assay conjugate, thus enhancing the interaction of nizing and in the assay hapten. KMe-type antibodies were able
the antibody with the target molecule and therefore increasing to perfectly tolerate site-heterologies, whereas KMa-type anti-
9
,24,25,29,30,39,40
the apparent affinity.
Accordingly, kresoxim- bodies just recognized the HRP–KMo heterologous tracer.
On the other hand, if adequate binding to the conjugate
methyl binding by the different antisera was tested with heter-
ologous HRP conjugates (Table 1). The number of working occurred, most heterologous combinations helped to consider-
combinations of immunoreagents was higher than expected, ably improve the antibody apparent affinity, mostly for those
7
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Fig. 4 Inhibition curves for the best pAb from each immunizing hapten as
evaluated by homologous competitive ELISA. Values are the mean of three inde-
pendent experiments.
Fig. 3 Synthetic sequences for the preparation of haptens KMe and KMo.
Table 1 IC50 (nM) values for kresoxim-methyl from assays using polyclonal
a
antibodies
Enzyme tracer
Fig. 5 Standard inhibition curves for antibody rKMc#1 using the HRP–KMc
homologous tracer (▲) and the HRP–KMe heterologous tracer (■). Values are
pAb
KMa
KMb
KMc
KMe
KMo
the mean of three independent experiments.
b
rKMa#1
rKMa#2
rKMb#1
rKMb#2
rKMc#1
rKMc#2
rKMe#1
rKMe#2
rKMo#1
rKMo#2
28
117
—
—
—
—
—
36
19
180
200
4
—
—
16
9
5
25
13
—
—
—
5
—
3
7
350
300
30
1000
12
nanomolar range were still obtained with antibodies raised by
the best immunizing haptens, KMo and KMe.
—
268
76
23
48
—
2
—
11
27
4
Monoclonal antibody characterization
20
9
—
—
—
In order to generate high-affinity kresoxim-methyl binders,
hybridomas were selected following a double screening com-
petitive procedure. By this strategy, a collection of novel mAbs
coming from mice immunized with each of the synthesized
haptens was attained. Each antibody was named after the
immunizing hapten, and numbered. All of the antibodies were
4
5
3
a
Values are the mean of three independent experiments. Homologous
b
combinations are indicated in bold-type letter.
.8 at the highest tested tracer concentration (300 ng mL ) and at the
lowest assayed antiserum dilution (1/3000).
Amax was lower than
−1
0
1
comprised of κ light chains and were of the IgG isotype,
pAbs that had rendered high IC50 values with the homologous except mAbs KMa#26 and KMb#322, which were of the IgG2b
conjugate. In fact, pAb rKMc#1 improved the IC50 value 90-fold isotype.
with the conjugate HRP–KMe (Fig. 5), and pAb rKMb#2
Affinity-purified mAbs were characterized versus the homo-
increased its apparent affinity 30-fold also with HRP–KMe. logous and all of the site-heterologous conjugates by checker-
Nevertheless, most combinations with IC50 values in the low board competitive direct ELISA. Half of the mAbs did not work
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Table 2 IC50 values (nM) for kresoxim-methyl from assays using monoclonal variety of sites was observed, thus proving a relationship
a
antibodies
between the structure of the immunizing hapten and the
apparent affinity of derived antibodies. It was seen that deriva-
tization at the a-ring of strobilurins triggered a moderate
Enzyme tracer
mAb
KMa
KMb
KMc
KMe
KMo immune response in terms of affinity to kresoxim-methyl, as
observed previously for other compounds of this family. In
contrast, high-affinity polyclonal and monoclonal antibodies
were obtained from immunogens with the linker at either end
of the (methoxyimino)acetate moiety, which possess higher
conformational freedom. Results from a hapten carrying the
linker at the para position of the characteristic o-cresol ring
b
KMa#26
KMa#29
KMb#19
KMb#32
KMb#44
KMb#322
KMc#35
KMe#14
KMe#18
KMe#26
KMe#211
KMo#111
KMo#114
KMo#117
—
—
—
—
2.02
3.41
0.76
—
—
—
—
—
—
—
—
—
—
0.60
—
0.66
—
—
—
—
—
—
—
0.68
—
0.97
—
1.77
1.90
—
—
2.13
—
—
—
—
—
—
0.78
—
1.72
1.05
—
—
—
—
—
0.60
—
—
—
—
—
—
—
—
(
hapten KMb) strongly differed between rabbit pAbs and
mouse mAbs. In spite of the drastic alteration introduced by
site-heterologies, pAbs are complex enough to be able to bind
such conjugates and to permit considerable sensitivity
improvements, mainly with moderate-affinity antibodies. In
contrast, few mAbs could tolerate such severe changes in
hapten structure, especially if the assay hapten held the linker
at an opposite site with respect to the immunizing hapten.
—
—
—
—
—
2.08
—
—
1.97
1.70
a
Values are the mean of three independent determinations and
correspond to an inhibition curve with an Amax value between 0.8 and
.5. Antibody concentration was either 300 or 1000 ng mL
−
1 b
1
.
A
max
−
1
values were lower than 0.8 at 300 ng mL tracer concentration and the
highest coating concentration (mAb at 1 µg mL− ).
1
Experimental
Chemicals and general procedures
satisfactorily, not even with the homologous enzyme tracer
(Table 2). Such behavior upon mAb immobilization has been
tert-Butyl hex-5-ynoate and tert-butyl 6-bromohexanoate were
prepared by esterification with isobutene of commercial hex-
5-ynoic acid and 6-bromohexanoic acid, respectively, following
a previously described procedure for the preparation of related
previously observed by several authors, and it is commonly
ascribed to conformational changes of antibodies resulting
4
1–44
from direct adsorption to hydrophobic surfaces.
As a fore-
most result, working mAbs exhibited IC50 values below 2 nM.
Interestingly, HRP conjugates of haptens KMe and KMo –
holding the linker at the (methoxyimino)acetate moiety – were
bound by both KMe- and KMo-type antibodies, indicating a
certain equivalency of these two adjacent derivatization sites. It
is worth noting that high-affinity mAbs were obtained from
hapten KMb. This result contrasts with the poor affinity exhibi-
ted by KMb-type pAbs, and raises the question of whether infor-
mation from rabbit antisera is a useful criterion to select
optimum haptens for hybridoma technology. In addition, anti-
body KMb#322 recognized all four heterologous tracers, yet
similar IC50 values were derived from all reagent combinations.
To gain further insights into the kresoxim-methyl binding
properties of the mAb collection, binders were also examined
in solution by competitive ELISA with immobilized OVA–
hapten conjugates. As shown in Table S1,† general remarks
were essentially identical; haptens KMo, KMe and KMb were
confirmed as the best mimics in order to generate high-affinity
anti-kresoxim-methyl mAbs, whereas haptens KMa and KMc
just elicited antibodies of moderate affinity. Moreover, the
widest tolerance to site-heterologies was again observed with
mAbs that had been triggered from hapten KMb.
1
5
tert-butyl esters. Other chemical reagents were purchased
from regular sources and used without purification. All oper-
ations involving air-sensitive reagents were performed under
an inert atmosphere of dry nitrogen or argon using syringe
and cannula techniques, oven-dried glassware, and freshly dis-
tilled and dried solvents. Organic solvents were dried and dis-
4
5
tilled before use according to standard procedures. Reactions
were followed by thin-layer chromatography using 0.25 mm
pre-coated silica gel plates and products were visualized by UV
light (254 nm) and aqueous ceric ammonium molybdate solu-
2 4
tion or 50% (v/v) concentrated H SO in water and then
heating with a high-temperature air dryer. Unless otherwise
stated, reaction mixtures were worked up by the addition of
water and extraction with the appropriate solvent, the organic
layer being washed with water and/or brine and dried using
anhydrous magnesium or sodium sulfate. Evaporation of the
solvent was performed under reduced pressure using a rotary
evaporator. Synthesized compounds were purified by flash
chromatography with
a silica gel column (Merck 60,
2
30–400 mesh). Melting points and spectra were recorded
3
8
as in previous studies. The most stable conformation of
kresoxim-methyl was calculated using CAChe WorkSystem Pro
software, version 7.5.0.85 (Fujitsu Ltd, Tokyo, Japan).
Kresoxim-methyl (methyl (E)-methoxyimino[α-(o-tolyloxy)-o-
tolyl]acetate, CAS Registry No. 143390-89-0, MW 313.35 g
mol ), Pestanal grade, was purchased from Fluka/Riedel-
Conclusions
−
1
A differential response of the immune system to a battery of de-Haën (Seelze, Germany). Horseradish peroxidase (HRP),
kresoxim-methyl regioisomeric haptens with the linker at a ovalbumin (OVA), N,N′-disuccinimidyl carbonate (DSC),
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Freund’s adjuvants, and o-phenylenediamine were purchased the solvent, using hexane–Et
2
O (from 9 : 1 to 8 : 2) as an
from Sigma/Aldrich (Madrid, Spain). Rabbit anti-mouse immu- eluent, afforded benzyl ether 3 (107.5 mg, 90%) as an oil.
noglobulins polyclonal antibody–peroxidase conjugate (RAM–
Synthesis of (E)-tert-butyl 6-(4-(2-methoxy-1-(methoxyimino)-2-
HRP) was from Dako (Glostrup, Denmark). Bovine serum oxoethyl)-3-((o-tolyloxy)methyl)phenyl)hex-5-ynoate (5). A solu-
albumin (BSA) fraction V and Hybridoma Fusion and Cloning tion of tert-butyl hex-5-ynoate (4, 155 mg, 0.920 mmol) in anhy-
Supplement (HFCS) were obtained from Roche Applied drous degassed DMF (1.0 mL) was added to a mixture of aryl
Science (Mannheim, Germany). P3-X63-Ag8.653 mouse plas- iodide 3 (101 mg, 0.230 mmol), CuI (1.3 mg, 0.007 mmol,
macytoma cell line was acquired from the European Collection 3% mol) and (Ph
of Cell Cultures (Wiltshire, UK). Polyethylene glycol Hybri-Max under nitrogen atmosphere. After the addition of anhydrous
PEG 1500), cell culture media (high-glucose Dulbecco’s Modi- Et N (0.5 mL), the reaction mixture, initially yellow that turned
3 2 2
P) PdCl (4.8 mg, 0.007 mmol, 3% mol)
(
3
fied Eagle’s Medium, DMEM), non-essential amino acid solu- deep-red after a few minutes, was stirred at room temperature
tion, 200 mM alanyl–glutamine solution, fetal bovine serum for 6 h, then poured into water, and worked up in the
(
FBS), and red blood cell lysing buffer were bought from usual manner using EtOAc to the extract. Work up as usual
Sigma/Aldrich (Madrid, Spain). Gentamicine, hypoxanthine– and chromatography of the crude product with hexane–Et
aminopterin–thymidine (HAT), and hypoxanthine–thymidine (7 : 3) as an eluent afforded alkyne 5 (94.7 mg, 86%) as a color-
HT) solutions were obtained from Gibco BRL (Paisley, Scot- less oil.
land). Culture plastic ware and Costar 96-well flat-bottom high-
Synthesis of (E)-tert-butyl 6-(4-(2-methoxy-1-(methoxyimino)-2-
binding polystyrene ELISA plates were from Corning (Corning, oxoethyl)-3-((o-tolyloxy)methyl)phenyl)hexanoate (6). A mixture
NY, USA). Sephadex G-25 HiTrap Desalting columns and of alkyne 5 (56.3 mg, 0.117) and Rh(PPh Cl (3.3 mg,
Sepharose HiTrap Protein G HP columns from GE-Healthcare 0.003 mmol) in anhydrous tetrahydrofuran (THF; 1 mL) was
Uppsala, Sweden) were used for conjugation and for antibody stirred under an atmosphere of hydrogen at 4 atm for 16 h.
2
O
(
3 3
)
(
purification, respectively, in the ÄKTA workstation. Ultraviolet– The reaction mixture was concentrated to dryness and the
visible spectra and ELISA absorbances were read using a obtained residue was purified by chromatography, using
PowerWave HT from BioTek Instruments (Winooski, VT, USA). hexane–EtOAc (9 : 1) as an eluent, to give the title compound 6
Microwells were washed with an ELx405 microplate washer, (48.2 mg, 85%) as a brownish oil.
also from BioTek Instruments. The immunoglobulin isotype
was determined using the Mouse MonoAb-ID kit from Invitro- 3-((o-tolyloxy)methyl)phenyl)hexanoic acid (hapten KMa). A solu-
gen (Carlsbad, CA, USA).
tion of tert-butyl ester 6 (48.2 mg, 0.099 mmol) in formic acid
Synthesis of (E)-6-(4-(2-methoxy-1-(methoxyimino)-2-oxoethyl)-
Composition, concentration, and pH of the employed (2 mL) was stirred at room temperature under nitrogen for 3 h.
buffers and solutions were as follows: (i) CB, 50 mM sodium Then, the reaction mixture was diluted with benzene and
carbonate–bicarbonate buffer, pH 9.6; (ii) PB, 100 mM sodium washed with cold water until pH 6–7 was attained, and dried
phosphate buffer, pH 7.4; (iii) PBS, 10 mM sodium phosphate over anhydrous Na
buffer, pH 7.4, with 140 mM NaCl; (iv) PBST, PBS containing residue left after evaporation of the solvent, using CHCl
.05% (v/v) Tween 20; (v) washing solution, 150 mM NaCl con- eluent, afforded hapten KMa (39.3 mg, 93%) as a colorless oil.
taining 0.05% (v/v) Tween 20; and (vi) substrate solution,
Synthesis of hapten KMb (Fig. 2)
Synthesis of (E)-methyl 2-(2-((4-iodo-2-methylphenoxy)methyl)-
5 mM sodium citrate and 62 mM sodium phosphate buffer, phenyl)-2-(methoxyimino)acetate (7). A 1.53 M solution of ICl in
SO
. Chromatographic purification of the
2
4
3
as an
0
−1
0
2 2
.012% (v/v) H O and 2 mg mL o-phenylenediamine in
2
pH 5.4.
CH
tion of kresoxim-methyl (537.5 mg, 1.720 mmol) in AcOH
3 mL) at room temperature. The resulting mixture was
stirred for 5 h, then poured into water and extracted with EtOAc.
The combined organic layers were washed successively with a
2 2
Cl (4.5 mL, 6.885 mmol) was added dropwise to a solu-
(
Syntheses of haptens
Five functionalized regioisomeric haptens of kresoxim-methyl
with a C-6 hydrocarbon spacer arm located at selected sites of
the molecular skeleton were synthesized (Fig. 1). Experimental
details of their preparation are given below, while a detailed
description of full spectroscopic data of all synthetic inter-
mediates is provided in the ESI (Fig. S1 to S5†).
10% aqueous solution of sodium thiosulfate, water and brine,
dried over anhydrous Na SO and concentrated, and the
2
4
obtained residue was purified by chromatography, using
hexane–EtOAc (8 : 2) as an eluent, to give the aryl iodide 7
(718.4 mg, 95%) as a white solid. Mp. 124–125 °C (crystallized
from hexane–benzene).
Synthesis of hapten KMa (Fig. 2)
Synthesis of (E)-methyl 2-(4-iodo-2-((o-tolyloxy)methyl)phenyl)-
Synthesis of (E)-tert-butyl 6-(4-((2-(2-methoxy-1-(methoxy-
2
(
-(methoxyimino)acetate (3). A mixture of benzyl bromide 1 imino)-2-oxoethyl)benzyl)oxy)-3-methylphenyl)hex-5-ynoate (8).
112 mg, 0.272 mmol), prepared in three steps from 1-iodo- The synthesis of this compound was performed following
3
0
-methylbenzene (see the ESI†), o-cresol (2, 28 µL, the same procedure reported for compound 5, starting from
.272 mmol), and anhydrous Cs CO (133 mg, 0.408 mmol) in aryl iodide 7 (529.8 mg, 1.21 mmol), alkyne 4 (811 mg,
2
3
dry N,N-dimethylformamide (DMF) (1.3 mL) was stirred under 4.84 mmol), CuI (6.9 mg, 0.040 mmol), Pd(PPh
nitrogen for 6 h, poured into water, and worked up using 0.040 mmol), DMF (3.9 mL), and Et N (2.9 mL), and 3 h of
EtOAc. Chromatography of the residue left after evaporation of reaction time. The crude product was purified by
) Cl (25.5 mg,
3 2 2
3
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chromatography, using hexane–EtOAc (from 9 : 1 to 8 : 2) as an the methyl ester moiety of kresoxim-methyl following the pro-
1
2
eluent, to give alkyne 8 (545.8 mg, 95%) as a yellowish oil.
Synthesis of (E)-tert-butyl 6-(4-((2-(2-methoxy-1-(methoxyimino)- 0.401 mmol) in dry DMF (13 mL) under nitrogen at room temp-
-oxoethyl)benzyl)oxy)-3-methylphenyl)hexanoate (9). Alkyne erature. The resulting mixture was stirred for 24 h, then poured
499.4 mg, 1.041 mmol) was hydrogenated as described above into water, and worked up in the usual manner by extracting
for the preparation of using Rh(PPh Cl (28.9 mg, with EtOAc. Purification of the crude product by chromato-
.031 mmol) and THF (9.5 mL). Purification of the crude product graphy, using hexane–Et O (from 8 : 2 to 7 : 3) as an eluent,
by chromatography with hexane–EtOAc (8 : 2) as an eluent gave afforded the title compound 17 (110 mg, 70%) as a colorless oil.
the title compound 9 (455.8 mg, 90%) as a semisolid.
Synthesis of (E)-6-(2-(methoxyimino)-2-(2-((o-tolyloxy)methyl)-
cedure previously described, and anhydrous K CO (54.4 mg,
2 3
2
(
8
6
3 3
)
0
2
Synthesis of (E)-6-(4-((2-(2-methoxy-1-(methoxyimino)-2-oxoethyl)- phenyl)acetoxy)hexanoic acid (hapten KMe). Hydrolysis of the
benzyl)oxy)-3-methylphenyl)hexanoic acid (hapten KMb). Hydro- tert-butyl ester group of 17 (36.9 mg, 0.078 mmol) with formic
lysis of the tert-butyl ester moiety of 9 (159 mg, 0.329 mmol) acid (1.6 mL) under the same conditions reported above for 6
with formic acid (6.8 mL) under the same conditions reported afforded, after chromatographic purification with CHCl
3
as an
above for 6, followed by purification by chromatography using eluent, hapten KMe (30.6 mg, 93%) as a colorless oil.
CHCl
3
as an eluent, afforded hapten KMb (127.8 mg, 91%) as a
Synthesis of hapten KMo (Fig. 3)
Preparation of (E)-tert-butyl 6-(((1-(2-(bromomethyl)phenyl)-2-
methoxy-2-oxoethylidene)amino)oxy)hexanoate (19). A mixture
of oxime 18 (synthesized following the procedure described by
slightly colored oil.
Synthesis of hapten KMc (Fig. 2)
Synthesis of (E)-methyl 2-(2-((2-iodophenoxy)methyl)phenyl)-2-
4
7,48
Li et al.)
16, 117 mg, 0.466 mmol), and anhydrous Cs
.699 mmol) in dry DMF (1.7 mL) was stirred under nitrogen
(90 mg, 0.466 mmol), tert-butyl 6-bromohexanoate
(
methoxyimino)acetate (12). This compound was prepared basi-
cally according to the procedure already described for com-
pound starting from benzyl bromide 10 (74.2 mg,
.259 mmol), 2-iodophenol (11, 30 µL, 0.259 mmol), and
Cs CO (125 mg, 0.388 mmol) in DMF (1.5 mL), and 4 h of
(
2
CO (228 mg,
3
0
6
4
6
for 4 h, poured into water, and worked up as usual using
EtOAc to extract. Chromatography of the crude product,
using hexane–EtOAc (9 : 1) as an eluent, afforded the corres-
ponding intermediate O-alkylated oxime (141 mg, 83%) as a
colorless oil.
0
2
3
reaction time. The crude product was purified by chromato-
graphy, using hexane–Et O (7 : 3) as an eluent, to give the title
compound 12 (72.6 mg, 90%) as a white solid. Mp. 87–89 °C
2
A
solution of the above obtained oxime (123 mg,
(crystallized from hexane–benzene).
0
0
.339 mmol), NBS (60.3 mg, 0.339 mmol), and AIBN (3.3 mg,
.020 mmol) in anhydrous benzene (1.5 mL) was degassed by
Synthesis of (E)-tert-butyl 6-(2-((2-(2-methoxy-1-(methoxy-
imino)-2-oxoethyl)benzyl)oxy)phenyl)hex-5-ynoate (13). The syn-
thesis of this compound was performed following the same
procedure that was described above for compound 5, starting
from aryl iodide 12 (78.0 mg, 0.239 mmol), alkyne 4 (48.0 mg,
nitrogen bubbling and ultrasound for 15 min and then heated
at reflux overnight. The reaction mixture was allowed to cool at
room temperature, diluted with EtOAc, and worked up as
usual to afford the crude product as a pale brown oil. Purifi-
0
.287 mmol), CuI (1.4 mg, 0.007 mmol), Pd(PPh
3 2 2
) Cl (5.0 mg,
cation by column chromatography, using hexane–Et O (from
0
.007 mmol), DMF (1.0 mL), and Et N (0.5 mL), and 2 h of
2
3
9
9
: 1 to 8 : 2) as an eluent, gave benzyl bromide 19 (137.8 mg,
2%; 76% overall yield from 18) as a brownish oil.
reaction time. The crude product was purified by chromato-
graphy, using hexane–Et O (8 : 2) as an eluent, to give 13
65.6 mg, 75%) as a colorless oil.
Synthesis of (E)-tert-butyl 6-(2-((2-(2-methoxy-1-(methoxy-
imino)-2-oxoethyl)benzyl)oxy)phenyl)hexanoate (14). Alkyne 13
65.6 mg, 0.180 mmol) was hydrogenated as described above for
the preparation of 6 using Rh(PPh ) Cl (5.0 mg, 0.005 mmol)
2
Synthesis of (E)-tert-butyl 6-(((2-methoxy-2-oxo-1-(2-((o-tolyl-
(
oxy)methyl)phenyl)ethylidene)amino)oxy)hexanoate
(20). This
compound was prepared following the procedure described
above for compound 3 but starting from benzyl bromide 19
(
(
54.7 mg, 0.124 mmol), o-cresol (12.8 µL, 0.124 mmol), and
Cs CO (60.6 mg, 0.186 mmol) in DMF (522 µL). The crude
product was purified by chromatography, using hexane–Et
9 : 12) as an eluent, to give the title compound as a colorless
oil (57.6 mg, 99%).
3
3
2
3
and THF (1.6 mL). Purification of the crude product by chrom-
atography with hexane–Et O (from 9 : 1 to 8 : 2) as an eluent
gave the title compound 14 (60.8 mg, 91%) as a colorless oil.
Synthesis of (E)-6-(2-((2-(2-methoxy-1-(methoxyimino)-2-
2
O
2
(
Synthesis of (E)-6-(((2-methoxy-2-oxo-1-(2-((o-tolyloxy)methyl)-
phenyl)ethylidene)amino)oxy)hexanoic acid (hapten KMo).
Hydrolysis of the tert-butyl ester moiety of 20 (115.2 mg,
oxoethyl)benzyl)oxy)phenyl)hexanoic acid (hapten KMc). Hydro-
lysis of the tert-butyl ester group of 14 (60.8 mg, 0.165 mmol)
with formic acid (3.4 mL) under the same conditions reported
above for 6 afforded, after chromatographic purification with
CHCl as an eluent, hapten KMc (46.5 mg, 90%) as a white
3
solid. Mp. 86–89 °C (crystallized from hexane).
0.246 mmol) with formic acid (5.0 mL) was carried out under
the same conditions reported above for 6, and after chromato-
graphic purification with CHCl3 as an eluent, hapten KMo
(
91.4 mg, 90%) was obtained as a colorless oil.
Synthesis of hapten KMe (Fig. 3)
Synthesis of (E)-tert-butyl 6-(2-(methoxyimino)-2-(2-((o-tolyloxy)-
methyl)phenyl)acetoxy)hexanoate (17). tert-Butyl 6-bromohexa-
Preparation of hapten active esters
noate (16, 100.6 mg, 0.401 mmol) was added to a mixture of The synthesized haptens were activated by formation of the
acid 15 (100 mg, 0.334 mmol), obtained by basic hydrolysis of corresponding N-hydroxysuccinimidyl ester (NHS ester) using
7
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Paper
DSC. Anhydrous Et
to a solution of the hapten (0.1 mmol) and DSC (26 mg,
.13 mmol, 1.3 equiv.) in dry acetonitrile (1 mL) under nitro-
3
N (54 μL, 0.38 mmol, 3.8 equiv.) was added Preparation of bioconjugates
For immunogen preparation, 100 µL of a 50 mM purified acti-
vated hapten (NHS ester) solution in DMF was added to a
0
gen at 0 °C. The reaction mixture was stirred at room temp-
−1
1
mL BSA solution (15 mg mL ) in CB. OVA conjugates were
erature overnight, then diluted with CHCl , washed with a
3
prepared by the addition of 100 µL of a 30 mM purified acti-
1
0% aqueous solution of NaHCO
anhydrous Na SO Chromatographic purification of the
residue that was left after evaporation of the solvent, using
CHCl as an eluent, afforded the NHS esters in good yield
3
and brine, and dried over
vated hapten solution in DMF over 2 mL of protein solution
2
4
.
−1
(
15 mg mL ) in CB. Likewise, enzyme tracers were prepared
by adding 100 µL of a 10 mM purified hapten-NHS ester solu-
tion in DMF over a 1 mL HRP solution (2.2 mg mL ) in CB.
3
−1
1
(
(
70–99%) and high purity, as shown by H NMR spectroscopy
copies of original spectra are included in the ESI†).
Mixtures were incubated for 4 h at room temperature in amber
glass vials and conjugates were purified by size-exclusion
chromatography using PB as an eluent. Hapten-to-protein
molar ratios were calculated from absorbance values of the
carrier before and after conjugation. BSA and OVA conjugates
were stored frozen, whereas HRP tracers were kept at 4 °C with
1
KMa-NHS ester: (88% yield). H NMR (CDCl
.37 (1H, br s, H-2′), 7.20 (1H, dd, J = 7.8, 1.5 Hz, H-6′), 7.14–7.07
3H, m, H-5′, H-3″ and H-5″), 6.85 (1H, br dd, J = 7.5, 7.5 Hz, H-4″),
.78 (1H, br d, J = 8.1 Hz, H-6″), 4.93 (2H, s, OCH ), 4.02 (3H, s,
NOCH ), 3.81 (3H, s, CO CH ), 2.82 (4H, br s, OCCH CH CO),
.67 (2H, t, J = 7.5 Hz, H-6), 2.59 (2H, t, J = 7.5 Hz, H-2), 2.24 (3H,
s, CH ), 1.78 and 1.69 (2H each, two partially overlapped quint,
J = 7.5 Hz, H-5 and H-3), 1.45 (2H, m, H-4).
3
, 300 MHz) δ
7
(
6
2
3
2
3
2
2
0.5% (w/v) BSA and 0.01% (w/v) thimerosal.
2
3
Generation of polyclonal antibodies
1
KMb-NHS ester: (70% yield). H NMR (C D , 300 MHz) 7.45
6
6
Animal manipulation was performed in compliance with the
laws and guidelines of the Spanish Ministry of Agriculture,
Fisheries, and Food and according to European Directive 2010/
(1H, dd, J = 7.4, 1.3 Hz, H-3″), 7.16 (1H, signal hidden by
solvent peak, H-6″), 7.08 (1H, ddd, J = 7.4, 7.4, 1.7 Hz, H-4″),
7
6
5
2
7
.02 (1H, ddd, J = 7.4, 7.4, 1.3 Hz, H-5″), 6.89 (1H, br s, H-2′),
.83 (1H, dd, J = 8.3, 1.9 Hz, H-6′), 6.72 (1H, d, J = 8.3 Hz, H-5′),
6
3 concerning protection of animals used for scientific pur-
poses. Two antisera were generated with each immunogen
from two 2 kg female New Zealand white rabbits, which were
subcutaneously immunized with 0.3 mg of BSA–hapten conju-
gate in 1 mL of a 1 : 1 mixture of PB and complete Freund’s
adjuvant. At 21 day intervals, animals were boosted with a 1 : 1
emulsion between incomplete Freund’s adjuvant and a PB
solution containing 0.3 mg of the same immunogen. Ten days
after the fourth injection, whole blood was collected from the
ear vein of the rabbits by intracardiac puncture. Blood samples
were allowed to coagulate overnight at 4 °C; then, the serum
was separated by centrifugation and immunoglobulins were
precipitated with 1 volume of saturated ammonium sulfate
solution. This procedure was repeated and the precipitates
were stored at 4 °C.
.02 (2H, s, OCH
2 3 2 3
), 3.61 (3H, s, NOCH ), 3.36 (3H, s, CO CH ),
,37 (5H, s and t overlapped, CH and H-6), 2.11 (2H, t, J =
3
2 2
.4 Hz, H-2), 1.7–1.5 (4H, m, OCCH CH CO), 1.5–1.3 (4H, two
partially overlapped quint, J = 7.7 Hz, H-5 and H-3), 1.12
(2H, m, H-4).
1
KMc-NHS ester: (98% yield). H NMR (CDCl
3
, 300 MHz) δ
7
1
.58 (1H, dd, J = 7.6, 1.5 Hz, H-3″), 7.45 (1H, ddd, J = 7.6, 7.6,
.5 Hz, H-4″), 7.38 (1H, ddd, J = 7.6, 7.6, 1.5 Hz, H-5″), 7.19
(1H, dd, J = 7.6, 1.5 Hz, H-6″), 6.88 (1H, ddd, J = 7.5, 7.5,
1
.2 Hz, H-5′), 6.77 (1H, d, J = 8.1 Hz, H-3′), 4.95 (2H, s, OCH
2
),
4
.04 (3H, s, NOCH ), 3.84 (3H, s, CO CH ), 2.83 (4H, br s,
3
2
3
2 2
OCCH CH CO), 2.66 (2H, t, J = 7.5 Hz, H-6), 2.57 (2H, t, J =
7
.5 Hz, H-2), 1.77 (2H, quint, J = 7.5 Hz, H-5), 1.64 (2H, quint,
J = 7.5 Hz, H-3), 1.47 (2H, m, H-4).
1
KMe–NHS ester: (98% yield). H NMR (CDCl
3
, 300 MHz) δ
Generation of monoclonal antibodies
7
1
.57 (1H, br d, J = 7.5 Hz, H-6′), 7.44 (1H, ddd, J = 7.5, 7.5,
.5 Hz, H-5′), 7.38 (1H, ddd, J = 7.5, 7.5, 1.4 Hz, H-4′), 7.21 (1H, A set of four BALB/c female mice (8–10 weeks old) was immu-
dd, J = 7.5, 1.5 Hz, H-3′), 7.15–1.07 (2H, m, H-3″ and H-5″), nized by intraperitoneal injections with each BSA–hapten
4
9
6
8
4
.86 (1H, ddd, J = 7.5, 7.5, 1.0 Hz, H-4″), 6.77 (1H, br d, J = conjugate following standard protocols. Briefly, the immu-
.0 Hz, H-6″), 4.95 (2H, s, OCH ), 4.21 (2H, t, J = 6.8 Hz, H-6), nization schedule consisted of four injections given at three
.03 (3H, s, NOCH ), 2.81 (4H, br s, OCCH CH CO), 2.52 (2H, week intervals. Each mouse received 100 µg of conjugate in
3 2 2
2
t, J = 7.5 Hz, H-2), 2.25 (3H, s, CH
3
), 1.66 (4H, m, H-3 and H-5), 200 µL of a 1 : 1 mixture of PBS and Freund’s adjuvant (com-
1
.37 (2H, m, H-4).
KMo–NHS ester: (98% yield). H NMR (CDCl
plete for the first dose and incomplete for the second and
, 300 MHz) δ third boosts). The fourth injection was done without adjuvant
1
3
7
1
.57 (1H, br d, J = 7.5 Hz, H-6′), 7.44 (1H, ddd, J = 7.5, 7.5, four days before mouse splenocytes were obtained and fused
.5 Hz, H-5′), 7.38 (1H, ddd, J = 7.5, 7.5, 1.5 Hz, H-4′), 7.19 (1H, with myelomas. Cell fusion was performed at a 4 : 1 ratio using
dd, J = 7.5, 1.2 Hz, H-3′), 7.11 (2H, m, H-3″ and H-5″), 6.85 (1H, 1 mL of PEG 1500 as the fusing agent. Fused cells were distrib-
5
ddd, J = 7.2, 7.2, 0.9 HZ, H-4″), 6.76 (1H, br d, J = 7.8 Hz, H-6″), uted in 96-well culture plates at 1.5 × 10 lymphocytes per well
4
.95 (2H, s, OCH ), 4.24 (2H, t, J = 6.9 Hz, H-6), 3.81 (3H, s, in 100 µL of DMEM containing 15% (v/v) FBS. Twenty-four
2
CO
H-2), 2.24 (3H, s, CH
.8 Hz, H-3 and H-5), 1.37 (2H, m, H-4).
2
CH
3
), 2.81 (4H, br s, COCH
), 1.66 (4H, two overlapped quint, J = plemented with HAT) with 20% (v/v) FBS and 1% (v/v) HFCS
was added to each well.
2 2
CH CO), 2.50 (2H, t, J = 7.5 Hz, hours after plating, 100 µL of selection medium (DMEM sup-
3
7
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Twelve days after cell fusion, hybridoma culture super- was defined as the antiserum dilution that was required in
natants were assayed following a sequential double-screening order to achieve both an Amax around 1.0 and a minimum IC50
process. First, cell cultures were screened by differential indir- value in homologous assays.
−
1
ect competitive ELISA with plates coated at 1.0 mg mL OVA–
5
0
hapten conjugate, as described by Abad et al. Briefly, each
culture supernatant was analyzed in parallel with and without
a competitor (100 nM kresoxim-methyl), and the obtained
Acknowledgements
absorbances were compared. The ratio between those assay This work was supported by the Spanish Ministerio de Ciencia e
signals was used as the criterion for the identification of the Innovación (MICINN) (AGL2006-12750-C02-01/02/ALI and
best clones that secreted antibodies recognizing not only the AGL2009-12940-C02-01/02/ALI) and cofinanced by FEDER
immobilized antigen (positive result) but also the target mole- funds. R.L.-M. was hired by MICINN under a predoctoral
cule; i.e., kresoxim-methyl (competitive result). In a second FPI grant associated to the above project. J.V.M. was hired by
screening assay, culture supernatants from positive and com- CSIC with a postdoctoral contract under the Ramón y Cajal
petitive wells, and also from those wells that afforded saturated program, cofinanced by MICINN and by the European Social
signals in the first screening experiment, were re-evaluated by Fund (ESF). We thank Ana Izquierdo-Gil and Laura López-
checkerboard indirect competitive ELISA as described by Mer- Sánchez for their excellent technical assistance. Limited
1
5
cader et al. Serial dilutions of the supernatant were assayed amounts of the described immunoreagents are available upon
in the presence of kresoxim-methyl at different concentrations request.
(0, 10, and 100 nM), using ELISA plates that had been coated
with the homologous OVA–hapten conjugate at 0.1 and 1.0 µg
−
1
mL . Selected hybridomas were cloned twice by limiting
dilution in cloning medium (DMEM containing 20% (v/v) FBS
and supplemented with HT and 1% (v/v) HFCS). Finally, stable
antibody-producing clones were expanded and cryopreserved
in liquid nitrogen. Antibodies were purified from late station-
ary-phase culture supernatants by salting out with ammonium
sulfate and subsequent affinity chromatography, and stored at
Notes and references
1 T. Anke, F. Oberwinkler, W. Steglich and G. Schramm,
J. Antibiot., 1977, 30, 806–810.
2 T. Anke, Can. J. Bot., 1995, 73, S940–S945.
3 D. W. Bartlett, J. M. Clough, J. R. Godwin, A. A. Hall,
M. Hamer and B. Parr-Dobrzanski, Pest. Manag. Sci., 2002,
58, 649–662.
4
°C as ammonium sulfate precipitates.
4
Preparation of new (phenoxymethyl)phenylglyoxylate
oxime ethers as fungicides, German Pat., DE3917352A1,
Competitive direct ELISAs
1
990.
Immunoassays were performed using the competitive ELISA
method with immobilized anti-kresoxim-methyl antibody.
Briefly, plates were coated with 100 µL per well of antibody
dilution in CB by overnight incubation at room temperature.
After each incubation step, microwells were washed four times
with washing solution. The competitive reaction was run by
the addition of 50 µL per well of analyte in PBS plus 50 µL per
well of HRP–hapten solution in PBST, and incubation during
5
Process for the preparation of arylacetic ester derivatives
via palladium-catalyzed cross coupling reaction, US Pat., 5
726 343, 1998.
6
7
8
9
M. P. Marco, S. Gee and B. D. Hammock, TrAC, Trends Anal.
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N. A. Lee and I. R. Kennedy, J. AOAC Int., 2001, 84, 1393–
1
h at room temperature. A signal was produced with 100 µL
per well of freshly prepared enzyme substrate solution. After
0 min at room temperature, enzyme activity was stopped
1406.
G. Shan, C. Lipton, S. J. Gee and B. D. Hammock, in Hand-
book of Residue Analytical Methods for Agrochemicals, ed.
Philip W. Lee, John Wiley & Sons, Ltd., Chichester, 2002,
pp. 623–679.
1
upon adding 100 µL per well of 2.5 M sulfuric acid. Absor-
bances were immediately read at 492 nm with a reference wave-
length at 650 nm.
1
0 Z. L. Xu, Y. D. Shen, R. C. Beier, J. Y. Yang, H. T. Lei,
H. Wang and Y. M. Sun, Anal. Chim. Acta, 2009, 647, 125–
Standard curves and data analysis
136.
Standard curves were set up from 10−2 µg L−1 to 10 µg L by 11 J. V. Mercader, C. Suárez-Pantaleón, C. Agulló, A. Abad-
4
−1
1
0-fold serial dilution in PBS. A blank (no kresoxim-methyl)
Somovilla and A. Abad-Fuentes, J. Agric. Food Chem., 2008,
56, 2581–2588.
was also included. Experimental values were fitted to a four-
parameter logistic equation using the SigmaPlot software 12 J. V. Mercader, C. Suárez-Pantaleón, C. Agulló, A. Abad-
package from SPSS Inc. (Chicago, IL, USA). Antibody affinity
was estimated as the concentration of kresoxim-methyl at the
Somovilla and A. Abad-Fuentes, J. Agric. Food Chem., 2008,
56, 1545–1552.
midpoint of the fitted curve, typically corresponding to a 50% 13 Z. L. Xu, H. Wang, Y. D. Shen, M. Nichkova, H. T. Lei,
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R. C. Beier, W. X. Zheng, J. Y. Yang, Z. G. She and
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