In order to develop immunoassays for pesticides detection, this work describes the choice of three different haptens that present structural similarity to benzimidazole molecule, methods for coupling them with two carrier proteins in order to make them immunogenic, a protocol for immunization of laboratory rodents with hapten-carrier complexes, and the evaluation of the specific antibody responses against haptens using an in-house developed immunoassay. Three carbendazim (methyl 2-benzimidazole-carbamate) derivatives bearing different functional reactive groups (-NH2, -SH and -COOH), namely 2-(2-Aminoethyl) benzimidazole (AEB), 2-Mercaptobenzimidazole (2MB) and 2-Benzimidazole propionic acid (BPA), were coupled to keyhole limpet haemocyanin (KLH) and bovine serum albumin (BSA), respectively, mixed with immuno-adjuvants, and injected four times into Balb/C mice and Wistar rats for induction of specific immune responses. All three chemicals elicited a specific but weak antibodies response upon immunization with hapten-KLH complexes, followed by serological testing by indirect ELISA using hapten-BSA complexes, and showed detectable differences in antibody titers with regard to number of inoculations, hapten structure and animal species. Whereas the AEB-KLH complex was the strongest, 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, immunogenicity, antibodies

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.


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 ( that offers details of over 7,250 possible immunogenic haptens and a percentage hierarchy of their 2-D similarity compared with the target structure (G”nther et al, 2007). We also accessed the HaptenDB (, 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 – NH2, -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 ‘NH2 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 (” 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 ”g protein) adsorbed on immuno-adjuvants – [Al(PO4)3] 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 4oC 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 37oC. 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 37oC) and washing (4 times), the color reaction was developed with SigmaFast OPD (Sigma-Aldrich) according to the manufacturers instructions, for 15-30 min at 37oC, and absorbance was measured with a plate reader (Infinite F200, Tecan Austria GmbH) at ” = 450 nm.


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 (G”nther 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 ”-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 spectra 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).

Figure 2. Antibody responses in Balb/C mice immunized against 2-(2-Aminoethyl) benzimidazole (AEB). 1st bleeding was done after two inoculations and the 2nd 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 results are presented as the mean optical density (O.D.) by ELISA 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 2nd 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 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 2nd 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 2nd 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 = 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 followed the immunization protocol with KLH-AEB complex via s.c. route 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 immunogenic hapten close to the ideal (Goodrow and Hammock, 1998; Tong et al, 2007; Song et al, 2010; Goel, 2013). With this 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 (-NH2, -COOH, -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 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 carbendazim by comparison to AEB (65.75% 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 also possible that the hapten chemical structures were not properly exposed for recognition by the immune system cells. As a consequence, there were less B-cell epitopes available for processing that ultimately led to a low antibody response. On the other 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, screening and cloning (Chiarella and Fazio, 2008).
Currently, we have ongoing experiments for getting monoclonal antibodies recognizing these haptens, and which can then be used in immunoassays development for detection of benzimidazole pesticide residues in food and feed.

Table 1. Five hapten candidates from top 30 structures most 2D-similar to carbendazim and possible to be used as immunogens based on the information provided by the SuperHapten database (
Name Structure ID 2D-Similarity
methyl 2-benzimidazolecarbamate
(carbendazim) 2426 100.00

2-aminobenzimidazole 2309

2-amino-5-(propylthio)benzimidazole 2302

2-mercaptobenzimidazole 10107

4`-hydroxyfenbendazole 2306


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.

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.

*** Commission Regulation (EC) No 149/2008 of 29 January 2008 amending Regulation (EC) No 396/2005 of the European Parliament and of the Council by establishing Annexes II, III and IV setting maximum residue levels for products covered by Annex I thereto
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