USE OF HAPTEN-CARRIER COMPLEXES FOR BENZIMIDAZOLE PESTICIDES IMMUNOASSAYS DEVELOPMENT
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 …
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