Synthesis of Haptens of Organophosphorus Pesticides and Development of Enzyme-linked Immunosorbent Assays for Parathion-methyl

Department of Biochemistry and Institute of Natural Sciences, Yeungnam University, Gyongsan 712-749, South Korea
Analytica Chimica Acta (Impact Factor: 4.51). 09/2003; 493(1):47-62. DOI: 10.1016/S0003-2670(03)00793-1


A simple synthetic method for haptens of organophosphorus (OP) pesticides with a spacer arm (aminocarboxylic acid) attached at the pesticide thiophosphate group was developed. While the previous synthetic approach for this type of haptens requires seven steps, the present method involves only two steps. Using this method, four haptens of the OP insecticide parathion-methyl were synthesized. Rabbits were immunized with either one of the two haptens coupled to bovine serum albumin for production of polyclonal antibodies. Using the serum with the highest specificity, an antigen-coated ELISA was developed, which showed an IC50 of 6.4 ng/ml with a detection limit of 0.2 ng/ml. An antibody-coated ELISA using an enzyme tracer was also developed, which showed an IC50 of 3.5 ng/ml with a detection limit of 0.3 ng/ml. The antibodies showed negligible cross-reactivity with other OP pesticides tested except with the insecticides parathion and paraoxon only in the antigen-coated ELISA.

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    • "0.3 Â 10 À3 mM) of BSA were dissolved in 2 mL of borate buffer (0.2 M, pH 8.7) to which 0.4 mL of DMF was added. A solution of an active ester (0.018 mM) in 0.1 mL of DMF was then added to the stirred protein solution, and stirring was continued for one day at 4 °C (Kim et al., 2003; Liu et al., 2007). 3.6. "
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    ABSTRACT: Agent orange herbicides, organophosphate and triazinic pesticides analysis in olive oil and industrial oil mill waste effluents using new organic phase immunosensors a b s t r a c t New immunosensors working in organic solvent mixtures (OPIEs) for the analysis of traces of different pesticides (triazinic, organophosphates and chlorurates) present in hydrophobic matrices such as olive oil were developed and tested. A Clark electrode was used as transducer and peroxidase enzyme as mar-ker. The competitive process took place in a chloroform–hexane 50% (V/V) mixture, while the subsequent enzymatic final measurement was performed in decane and using tert-butylhydroperoxide as substrate of the enzymatic reaction. A linear response of between about 10 nM and 5.0 lM was usually obtained in the presence of olive oil. Recovery tests were carried out in commercial or artisanal extra virgin olive oil. Traces of pesticides were also checked in the oily matrix, in pomace and mill wastewaters from an indus-trial oil mill. Immunosensors show good selectivity and satisfactory precision and recovery tests per-formed in olive oil gave excellent results.
    Full-text · Article · Feb 2015 · Food Chemistry
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    • "However, OP pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment [1]. Additionally, there is increasing concern over food and environmental contamination resulting from the overuse of pesticides [2]. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment. "
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    ABSTRACT: Organophosphorus (OP) pesticides are considered hazardous substances because of their high toxicity to nontarget species and their persistence in the environment and agricultural products. Therefore, it is important to develop a rapid, sensitive, and economical method for detecting OP pesticides and their residues in food and the environment. A broad, selective monoclonal antibody (MAb) for organophosphorus pesticides was produced. Based on the MAb, an enzyme linked immunosorbent assay (ELISA) and an immunochromatography assay (ICA) for detecting OP pesticides in different agricultural products were developed using a binding inhibition format on microtiter plates and a membrane strip, respectively. Under the optimized conditions, the IC(50) values of the ELISA ranged from 3.7 to 162.2 ng mL(-1) for the 8 OP pesticides. The matrix interferences of Apple, Chinese cabbage, and greengrocery were removed by 40-fold dilution, the recoveries from spiked samples ranged from 79.1% to 118.1%. The IC(50) values of ICA for the 8 OP pesticides ranged from 11.8 to 470.4 ng mL(-1). The matrix interference was removed from the Chinese cabbage and Apple samples with 5-fold dilution, and the interference was removed from the greengrocery samples with 20-fold dilution. The recoveries from the spiked samples ranged between 70.6 and 131.9%. The established ELISA and ICA were specific selectivity for the 8 OP pesticides. The established ELISA is a sensitive screening method for the detection of OP pesticides, but the ELISA detection method depends on a laboratory platform and requires a relative long assay time and several steps operation. The established ICA is very useful as a screening method for the quantitative, semi-quantitative or qualitative detection of OP pesticides in agricultural products, and it has advantages over ELISA methods with regard to factors such as the testing procedure, testing time, and matrix interferences, among others.
    Full-text · Article · Dec 2012 · PLoS ONE
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    • "Accordingly, the generated antibodies generally show low specificity and high degree of cross-reactivity with other similarly structured molecules. Design of a hapten and a suitable carrier protein are therefore critical to ensure successful generation of antibodies having the desired specificity for therapeutic monitoring and immunodiagnostic studies (Kim, 2003). Here, we report first time a novel method for synthesis of hapten using a bacterial isolate for the preparation of hapten–protein conjugate for specific antibodies generation against commonly used herbicide diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea). "
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    ABSTRACT: A Gram-positive, Micrococcus sp. strain PS-1 isolated from diuron storage site was studied for its capability of biotransformation of phenylurea herbicide diuron to a secondary metabolite, 1-(3,4-dichlorophenyl)urea (DCPU) for bioconjugation and antibody development applications. The metabolite formed associated with profound changes in bacterial cell morphology demonstrated increase in the degradation kinetics of diuron in presence of small quantity of a surfactant. The synthesized metabolite identified by chromatographic and mass spectrometry techniques was conjugated with carrier protein, and used as an immunogen for antibodies production. The generated antibody was highly specific, demonstrating excellent sensitivity against diuron. The antibody was used as receptor molecules in standard fluorescence immunoassay (FIA) format showing detection limit of 0.01 ng/mL in the optimum working concentration range of diuron with good signal precision (∼2%). The study presented first time the degradation pathway of herbicide by specific microorganism to synthesize hapten for bioconjugation and immunoassay development.
    Full-text · Article · Oct 2010 · Bioresource Technology
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