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USE OF HAPTEN-CARRIER COMPLEXES FOR BENZIMIDAZOLE
PESTICIDES IMMUNOASSAYS DEVELOPMENT

Veronica TANASA 1,2 , Radu I. TANASA 3, Madalina DOLTU 2,
Gabriela HRISTEA 4, Narcisa BABEANU 1

1University of Agronomical Sciences and Veterinary M edicine in Bucharest,
59 Marasti Blvd, District 1, Bucharest, 011464, Rom ania
2Institute of Research and Development for Industria lization and Marketing of Horticultural
Products – HORTING, 1A Intrarea Binelui, District 4 , Bucharest, 042159, Romania
3National Institute of Research “Cantacuzino”, 103 S plaiul Independentei,
District 5, 050096, Bucharest, Romania
4National Institute of Research and Development in Electrical Engineering ICPE-CA,
313 Splaiul Unirii, District 3, 030138, Bucharest, Romania

Corresponding author email: [anonimizat]

Abstract

In order to develop immunoassays for pesticides de tection, this work describes the choice of three different haptens
that present structural similarity to benzimidazole molecule, methods for coupling them with two carri er proteins in
order to make them immunogenic, a protocol for imm unization of laboratory rodents with hapten-carrier complexes,
and the evaluation of the specific antibody respons es against haptens using an in-house developed immu noassay. Three
carbendazim (methyl 2-benzimidazole-carbamate) der ivatives bearing different functional reactive grou ps (-NH 2, -SH
and -COOH), namely 2-(2-Aminoethyl) benzimidazole (AEB), 2-Mercaptobenzimidazole (2MB) and 2-Benzimidazole
propionic acid (BPA), were coupled to keyhole limpe t haemocyanin (KLH) and bovine serum albumin (BSA),
respectively, mixed with immuno-adjuvants, and inje cted four times into Balb/C mice and Wistar rats fo r induction of
specific immune responses. All three chemicals elicited a specific but weak a ntibodies response upon immunization
with hapten-KLH complexes, followed by serologica l testing by indirect ELISA using hapten-BSA comple xes, and
showed detectable differences in antibody titers with regard to number of inoculations, hapten str ucture and animal
species. Whereas the AEB-KLH complex was the strong est, the 2MB-KLH complex was the weakest immunogen in
mice. However, the best animal responders allow the application of technologies for getting monoclonal antibodies
against benzimidazoles, which can then be used for immunoassays development.
Key words : pesticides, hapten-carrier, benzimidazole, immuno genicity, antibodies

INTRODUCTION

Pesticides play a major role in improving
agricultural production through control of pest
populations such as insects, weeds, and plant
diseases. Unfortunately, the toxicological
properties of pesticides provide a potential
risks to humans, to the environment, and to
non-target organisms that might be
inadvertently exposed to such chemicals as
well. In particular, pesticides pose risks to
agricultural workers involved in mixing,
loading, and application of pesticides, as well
as to those who perform works in agricultural
settings where pesticides have been applied
(Winter, 2012). Despite their merits, pesticides
are considered to be among of the most dangerous environmental contaminants because
of their ability to accumulate and their long-
term effects on living organisms. The presence
of pesticides in the environment is
particularly hazardous, and exposure to
these pesticides leads to several health
problems that range from asthma attacks, skin
rashes, severe eye irritation and chronic
disorders to neurological diseases (Aragay et al,
2012; Schrenk, 2012). In the European Union,
the use of pesticides is strictly regulated and all
EU Member States apply the same evaluation
procedures and authorization criteria, in
order to place a plant protection product on
the market. In this respect the European Union
legislation has established a maximum residue
level (MRL) for food and feed of plant

and animal origin [Commission Regulations
(EC) 396/2005, amended by Commission
Regulations (EC) No. 149/2008] which is
updated as necessary (Keikothhaile and
Spanoghe, 2011). The identification and
quantification of pesticides are generally
based on gas chromatography – mass
spectrometry (GC-MS), liquid chromatography
– mass spectrometry (LC-MS), or high
performance liquid chromatography – mass
spectrometry (HPLC-MS) (Nunes and Barcelo,
1999). These methods permit precise and
accurate detection, and quantification of trace
levels of hundreds of these chemicals.
However, these conventional methods for
pesticides monitoring require multiple steps for
sample preparation and analysis, often
including derivatization, highly trained
personnel, expensive specialized equipment,
and are time consuming (Schrenk, 2012).
Immunoassays are based on the use of anti-
pesticide antibodies (Ab) as the specific
sensing element and that can provide
concentration-dependent signals. Such assays
appear to be appropriate for identification of a
single pesticide or, in some cases, small groups
of similar pesticides in food, feed and
environmental matrices, as they are rapid,
specific, sensitive and included in cost-
effective analytical devices. Furthermore, they
can be used and interpret in the field by
operators with minimal training and,
generally, do not require sophisticated
equipment to be accomplished. Therefore,
developing such immunoassays has gained
popularity during the recent years (Fan and He,
2011; Liu et al, 2013).
On the other hand pesticides, organic
compounds of molecular mass less than 1,000,
are usually non-immunogenic, and hence do
not elicit an immune response unless coupled
with some macromolecules such as proteins.
Therefore, it is necessary to modify these
chemicals – known as haptens – by coupling
them with macromolecules – known as
carriers – in order to make a stable carrier-
hapten complex. The carrier-hapten complexes
can then be used to generate antibodies against
pesticides (Dankwardt, 2000; Raman et al,
2002). Also, because of the small size of these
pesticide molecules, a suitable immunoassay technique must be employed for their
detection.
In this paper we report the preparation of
hapten-carrier immunogenic complexes derived
from benzimidazole pesticides and their use for
stimulation of antibody responses in laboratory
animals.

MATERIALS AND METHODS

a) Haptens selection strategy
We considered as a target structure the
pesticide carbendazim (methyl 2-
benzimidazole-carbamate), a well known and
extensively fungicide used in agriculture and
horticulture, and which has not longer been
approved for use in the European Union
starting with 2015. In order to select some
similar chemical structures that would be able
to be used as immunogens, we used the public
SuperHapten database
(http://bioinformatics.charite.de/superhapten/)
that offers details of over 7,250 possible
immunogenic haptens and a percentage
hierarchy of their 2-D similarity compared
with the target structure (Günther et al, 2007).
We also accessed the HaptenDB
(http://www.imtech.res.in/cgibin/haptendb/inde
x.html), a bioinformatic database that includes
similar information of over 1,080 haptens,
including pesticides (Singh et al, 2006). On the
other hand, there have been recently reported
results on the induction of a antibody responses
against carbendazim and similar compounds
using commercial chemical structures having –
NH 2, -COOH, – SH, -OH as functional groups
for conjugation with protein carriers (Moran et
al, 2002; Gough et al, 2011; Zikos et al, 2015).
Since customized synthesis of similar
compounds to a pesticide target is quite
expensive, and after collating all the
information provided by the previously-
described strategy, we have chosen to use the
following three commercially available
carbendazim derivatives:

1. 2-(2-Aminoethyl) benzimidazole (AEB)
(Sigma-Aldrich, 98% purity), which has –NH 2
as a functional reactive group;

2. 2-Mercaptobenzimidazole (2MB) (Sigma-
Aldrich, 98% purity), which has –SH as a
functional reactive group;

3. 2-Benzimidazole propionic acid (BPA)
(Sigma-Aldrich, 97% purity), which has –
COOH as a functional reactive group.

b) Haptens conjugation
Each hapten was conjugated to carrier protein
keyhole limpet haemocyanin (KLH; mcKLH –
a product of Thermo Scientific) and bovine
serum albumin (BSA; the product of Serva
Feinbiochemica GmbH &Co, or the Imject ®
BSA which is a product of Thermo Scientific),
following previously established conjugation
chemistry steps (Singh et al, 2004; Hermanson
2013).
The AEB-KLH and AEB-BSA complexes were
prepared through glutaraldehyde-mediated link
chemistry, using a previously-described
protocol (Hermanson, 2013).
The 2MB-KLH and 2MB-BSA complexes
were prepared using commercial carriers
(mcKLH and BSA Imject®, Thermo Scientific)
with sulfo-SMCC (succinimidyl 4- [N-
maleimidomethyl] cyclohexane-1-Carboxylate)
(Pierce, Thermo Scientific) as a
heterobifunctional linker, as directed by the
manufacturer.
The BPA-KLH and BPA-BSA complexes were
obtained using commercial kits (EDC Imject®
Carrier Protein Kits Thermo Scientific),
through EDC (1-ethyl-3- [3-
dimethylaminopropyl] carbodiimide
hydrochloride) – mediated chemistry, as
directed by the manufacturer.
We used a molar ratio of 1:40 and 1:900
(carrier:hapten) for preparation of BSA-hapten
complexes and KLH-hapten complexes,
respectively.
Hapten-carrier complex formation was
evaluated by UV-VIS spectrophotometry
(Abad et al, 1999), recording the spectra in the
regions of the maximum absorbance of the unconjugated and conjugated protein ( λ max =
275-280 nm) and hapten, respectively, or by
using 2,4,6-trinitrobenzene 1-sulfonic acid
(TNBS) reagent (Sashidhar et al, 1994).

c) Animals and immunizations
Immunizations were carried out using female
Balb/C mice (4 animals/group) and Wistar rats
(2 animals/group) of 6-8 weks of age. The
animals were reared in clean standard
environment, with food and water supply ad
libitum . The experimental protocol with
animals was performed in accordance with
relevant institutional and national guidelines
and regulations, and was approved by the
Ethics Committee of The National Research
Institute „Cantacuzino” (Application CE/
36/04.02.2015).
For induction of antibody responses against
haptens, the mice were inoculated with KLH-
hapten complexes only, via subcutaneous (s.c)
route first, and then with three booster
immunisations via intraperitoneally (i.p.) route,
every two weeks apart, with a combination of
hapten-carrier complex (30-100 µg protein)
adsorbed on immuno-adjuvants – [Al(PO 4)3]
plus Gerbu adjuvant MM (GERBU Biotechnik
GmbH, Heidelberg, Germany) – in a 0.05-0.2
ml final volume / animal. There were four
groups of mice used for immunization, of
which 3 groups were inoculated with each
KLH-hapten complex (KLH-AEB, KLH-2MB,
KLH-BPA) and another one was inoculated
with a mixture of all three complexes. The rats
(one group) were immunized with a mixture of
all KLH-hapten complexes + adjuvants, using
volumes of 0.2-0.5 ml/animal. Other negative
control groups of mice and rats were mock
immunized with KLH + adjuvants only.
All the animals were bled from the tail veins
before the immunization schedule first, and
then one week apart from the second (day 14)
and the forth injection (day 42). The serum was
separated from blood by centrifugation and
used for evaluation of the antibody response
against haptens by ELISA.

d) Hapten antibody response evaluation
Because KLH and BSA do not induce a
detectable cross-reactive immuno-response, the
BSA-hapten complexes were used as antigens
for in vitro evaluation of antibody responses

against haptens, by indirect ELISA. MaxiSorp
ELISA plates (Nunc, Roskilde, Denmark) were
coated overnight at 4 oC with the corresponding
BSA-hapten complex (5 ug/ml), in carbonate-
bicarbonate buffer (pH-9.6). After blocking
with 1% caseinate in phosphate-buffered saline
(PBS) and washing with PBS-Tween 20
(PBST, 4 times), twofold serial dilutions of the
sera (in PBS), starting from 1/10, were
incubated for 1 h at 37 oC. After washing (4
times), the plates were incubated for 1 hour
with either anti-mouse-IgG or anti-rat-IgG
peroxidase conjugated secondary antibodies
(SouthernBiotech, Birmingham, AL, U.S.A.),
diluted (1/1000) in PBS. After incubation (1
hour at 37 oC) and washing (4 times), the color
reaction was developed with SigmaFast OPD
(Sigma-Aldrich) according to the
manufacturers instructions, for 15-30 min at
37 oC, and absorbance was measured with a
plate reader (Infinite F200, Tecan Austria
GmbH) at λ = 450 nm.

RESULTS AND DISCUSSIONS

a) Haptens selection and conjugation
We found the hapten bioinformatics databases
very useful for rapid orientation and down-
narrowing of the screening, in order to find
suitable hapten candidates. Particularly,
SuperHapten Database offers 2-D / 3-D
structural details of possible immunogenic
haptens, their scientific and commercial
information, physicochemical properties, and a
percentage hierarchy of their 2-D similarity
compared with the target structure (Günther et
al, 2007). These information are very important
when choosing a suitable chemical structure
able to be coupled with carriers and then to
induce a suitable antibody response that can be
further exploited for the development of
reliable immunoassays.
In this way, in our experiments, we selected a
total of five preliminary candidates that have
similar structure to that of carbendazim and,
therefore, possibly to be used as immunogens
(Table 1). However, we decided to select the
final haptens (AEB, 2MB and BPA) after
further taking into account the previously
published results on this topic and of the
relevant trade information provided by the well known life science chemical substances
manufacturers, also.
By scanning the absorbance of proteins,
haptens and conjugates we found some subtle
deviations from the unconjugated proteins,
especially in the case of BSA-AEB complex
formation (Figure 1) but obvious changes in
absorbance spectra were not obtained with the
some other conjugates, in agreement with
another report (Gough et al, 2011). However,
we found evidence that conjugation had taken
place using TNBS reagent that strongly reacted
with the ε-amino groups of L-lysine present in
free carrier proteins, but less after hapten-
protein cross-linking (data not shown).

Figure 1. Evidence of a hapten-protein conjugation
through spectrophotometry. Overlapped UV-VIS spectr a
demostrate a shifting from the spectrum of the BSA
protein alone (in red), in comparison to BSA-AEB
complex, either before (in blue) and after dialysis (in
green).

By assuming that the molar absorptivity of
haptens was the same for free and conjugated
forms (Abad et al, 1999), apparent molar ratio
was estimated as ~10, in the case of BSA-
hapten complexes. We did not estimate this
ratio in the case of KLH-complexes but, we
assumed that because we used the same
protocols, and the KLH is a very large protein
(MW 4.5 × 10 5 to 1.3 × 10 7 ) with over 4,600
functional groups available for conjugation/
mole in comparison to BSA (MW 67,000) that
has over 100 such functional groups
(Hermanson, 2013), it was enough hapten
bound to carrier to induce an immune response.

b) Antibody responses against haptens
By ELISA, we detected antibodies that reacted
with the corresponding hapten, albeit of low
intensity, in all groups of mice, except the
negative control group (Figure 2, 3, 4 and 5). 04
123
200 350 250 300 Abs
Wavelength [nm]

Figure 2. Antibody responses in Balb/C mice
immunized against 2-(2-Aminoethyl) benzimidazole
(AEB). 1st bleeding was done after two inoculations and
the 2 nd bleeding was done after four inoculations. The
results are presented as the mean optical density ( O.D.)
by ELISA with standard deviation bars of n = 4
mice/group

Figure 3. Antibody responses in Balb/C mice
immunized against 2-Mercaptobenzimidazole (2MB).
1st bleeding was done after two inoculations and the 2nd
bleeding was done after four inoculations. The resu lts are
presented as the mean optical density (O.D.) by ELI SA
with standard deviation bars of n = 4 mice/group

Figure 4. Antibody response in Balb/C mice immunized
against 2-Benzimidazole propionic acid (BPA). 1st
bleeding was done after two inoculations and the 2 nd
bleeding was done after four inoculations. The res ults
are presented as the mean optical density (O.D.) by
ELISA with standard deviation bars of n = 4 mice/gr oup

Figure 5. Antibody responses in Balb/C mice
immunized against a mixture of AEB+2MB+BPA
haptens. 1st bleeding was done after two inoculations and
the 2 nd bleeding was done after four inoculations. The
results are presented as the mean optical density ( O.D.)
by ELISA with standard deviation bars of n = 4
mice/group

Furthermore, we clearly found an increased
response in antibodies, by indirect ELISA, after
the 4th inoculation relative to the 2nd
inoculation, that is relevant for the immune
maturation process that took place inside the
body after repeated antigenic stimulation. On
the other hand, there were differences in the
antibody response against each hapten, with
better results when used AEB-carrier and a
very low response against 2MB-carrier
complex, though potent adjuvants for B-cell
stimulation and differentiation were employed.
Even when used a mixture of all there haptens
we obtained the same poor results (Figure 5),
that is the general characteristic of the immune
responses against haptens.
The rats elicited a better antibody responses
against a mixture of all haptens (Figure 6) in
comparison to mice, the most probably due to
differences in immunoreactivity between these
animal species, a well known phenomenon.

Figure 6. Antibody responses in Wistar rats immunized
against a mixture of AEB+2MB+BPA haptens. 1st
bleeding was done after two inoculations and the 2 nd
bleeding was done after four inoculations. The resu lts are
presented as the mean optical density (O.D.) by ELI SA
with standard deviation bars of n = 2 rats/group

All immunogenic complexes were well
tolerated by the Balb/C mice, except KLH-

AEB that induced a moderate, nodular
dermatitis at the s.c. inoculation sites, but was
letal within 24-48 hours post- innoculation
when administered via i.p. route into an animal.
Therefore, we fo llowed the immunization
protocol with KLH-AEB complex via
only.
How can one explain the differences in
immunoreactivity to haptens within the same
species? It has been shown that small
molecules very often show low
immunogenicity that is mainly due to the rapid
breakdown of the molecule in vivo or clearance
via the renal pathway (Moran et al, 2002).
Therefore, both the hapten selection and the
choice of carriers have a qualitatively and
quantitatively influence on the immune
responses, including the secretion of antibodies.
Because of these reasons , some rules have been
established that likely would lead to make an
immuno genic hapten close to the ideal
(Goodrow and Hammock, 1998; Tong et al,
2007; Song et al, 2010; Goel , 2013)
regard, the hapten should ( i) have the structure,
conformation and physicochemical properties
as close to perfection as compared to the target
chemical structure(s); (ii) have in its structure
aromatic rings / hetero- aromatic rings /
branched radicals, and at least one reactive
functional group (-NH 2, -COOH, – OH,
attachment by covalent bonds to the carrier;
(iii) keep the original conformation after
Table 1. Five hapten candidates from top 30 structures
immunogens based on the
(http://bioinfo
Name
methyl 2-benzimidazolecarbamate
(carbendazim)
2-succinamidobenzimidazole
2-aminobenzimidazole AEB that induced a moderate, nodular
dermatitis at the s.c. inoculation sites, but was
innoculation
i.p. route into an animal.
llowed the immunization
via s.c. route
ow can one explain the differences in
within the same
It has been shown that small
molecules very often show low
due to the rapid
or clearance
via the renal pathway (Moran et al, 2002).
both the hapten selection and the
choice of carriers have a qualitatively and
quantitatively influence on the immune
the secretion of antibodies.
, some rules have been
established that likely would lead to make an
genic hapten close to the ideal
Hammock, 1998; Tong et al,
, 2013) . With this
i) have the structure,
conformation and physicochemical properties
as close to perfection as compared to the target
have in its structure
aromatic rings /
and at least one reactive
OH, -SH) for
attachment by covalent bonds to the carrier;
iii) keep the original conformation after coupling to a carrier and, if coupled to a carrier
molecule via a linking spacer, the latter
immunologically unresponsive.
Therefore, the very low antibody response
against 2MB can partially be explained by the
simpler structure of this chemical compound
and a less degree of similarity (61.00%) to
carbendazim by comparison to
similarity) (Table 1) and BPA. Another
possibility is that the linking reaction efficiency
was much lower for 2MB in comparison to
AEB and BPA, respectively. It is
that the hapten chemical structures were not
properly exposed for recognition
immune system cells. As a consequence
were less B- cell epitopes av
processing that ultimately led to a low antibody
response. On the o ther hand, even in the case
of poor antibody responses against haptens, it is
possible to isolate monoclonal antibodies with
the desired specificity by employing high
throughput strategies for fusion,
cloning (Chiarella and Fazio, 2008).
Currently, we have ongoing experiments for
getting monoclo nal antibodies recognizing
these haptens, and which can then be used in
immunoassays development for detection of
benzimidazole pesticide residues
feed.

from top 30 structures most 2D-similar to carbendazim and possible to be used as
based on the information provided by the SuperHapten database
http://bioinfo rmatics.charite.de/superhapten/)
Structure ID
2426

2425

2309

if coupled to a carrier
a linking spacer, the latter must be
immunologically unresponsive.
Therefore, the very low antibody response
against 2MB can partially be explained by the
simpler structure of this chemical compound
and a less degree of similarity (61.00%) to
to AEB (65.75%
1) and BPA. Another
possibility is that the linking reaction efficiency
was much lower for 2MB in comparison to
It is also possible
structures were not
for recognition by the
As a consequence , there
cell epitopes av ailable for
led to a low antibody
ther hand, even in the case
against haptens, it is
possible to isolate monoclonal antibodies with
employing high –
throughput strategies for fusion, screening and
cloning (Chiarella and Fazio, 2008).
Currently, we have ongoing experiments for
nal antibodies recognizing
these haptens, and which can then be used in
for detection of
enzimidazole pesticide residues in food and
and possible to be used as
database
2D-Similarity
100.00
79.89
65.75

2-amino-5-(propylthio)benzimidazole
2302

61.74
2-mercaptobenzimidazole
10107

61.00
4`-hydroxyfenbendazole
2306

60.36

CONCLUSIONS

1. In order to prepare immunogenic haptens for
developing antibodies against benzimidazole
pesticides, a working algorithm involving
checking of public bioinformatics databases
was applied for selection of similar chemical
structures to carbendazim (methyl 2-
benzimidazole-carbamate) and bearing
different reactive groups available for
conjugation to protein carriers.
2. Three commercial chemical compounds,
namely 2-(2-Aminoethyl) benzimidazole
(AEB), 2-Mercaptobenzimidazole (2MB) and
2-Benzimidazole propionic acid (PBA), were
coupled to carrier protein keyhole limpet
haemocyanin (KLH), mixed with immuno-
adjuvants, and injected four times into Balb/C
mice and Wistar rats, respectively.
3. All haptens induced a weak but specific
antibody response, as evidenced by an in-house
developed indirect ELISA, and with detectable
differences with regard to number of
inoculations, chemical structure and animal
species.
4. The AEB molecule induced the strongest
and the 2MB molecule induced the weakest
antibody responses in mice.
5. Our works showed evidence that further
inoculations are necessary in order to properly
stimulate the immune responses for generation
of monoclonal antibodies against
benzimidazoles.

ACKNOWLEDGEMENTS

This work was supported by the Romanian
Ministry of Education and Scientific Research –
Executive Agency for Higher Education,
Research, Development and Innovation
Funding (UEFISCDI), under the National
R&D&I Plan II – Partnering Program, Grant
PN II-PT-PCCA-2013-4-0128, Contract no.
147/2014 to G.H., R.I.T. and M.D. Some
expenditures for dissemination of results were
supported by the Doctoral School in
Engineering and Plant and Animal Resources
Management of the University of Agronomical
Sciences and Veterinary Medicine in
Bucharest, for V.T. and N.B.
All the authors declare no conflict of interest.

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