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This study was performed to demonstrate the importance of selecting an appropriate membrane when developing immunochromatographic assays (ICAs) for the sensitive detection of low-molecular weight compounds. Based on our findings, we propose a theoretical basis for selecting such a membrane. When eluting the sample solution for the competitive ICA using colloidal gold label for low-molecular analytes, the degree of binding inhibition is proportional to the collision frequency between the antibody-colloidal gold (Ab-CG) and analyte before Ab-CG binding to the capture antigen and a higher concentration of pesticides around the Ab-CG leads to a greater degree of inhibition. Therefore, we propose that the relative migration speed of the analyte and Ab-CG on the test strip is critically important for selecting a membrane in the development of sensitive competitive ICAs. We developed a novel method to estimate such a relative migration speed. We demonstrated the applicability of this proposal by using it to select an appropriate membrane for the development of an ICA of the pesticide diazinon., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Enzyme-linked immunosorbent assays (ELISAs) for the class-specific determination of organophosphorus (OP) pesticides were developed from monoclonal antibodies raised against haptens with the functional group common to OP pesticides. To develop antigen-coated, indirect, competitive ELISAs, four haptens with different spacer arm structures were used to prepare antibodies, while eight haptens were tested for use as coating antigens. A total of 32 ELISAs were developed with one selected as the most suitable one based on average IC(50) and % CV values. The chosen ELISA showed class-selective response to O,O-diethyl phosphorothioate and phosphorodithioate OP pesticides with negligible cross-reactivity to other types of pesticides. Average IC(50) and % CV values of this ELISA for the 12 OP pesticides were 89 ng/mL and 96%, respectively. Compared to ELISAs previously developed with the same objective, the current ELISA demonstrates better sensitivity based on much lower mean IC(50) values in addition to improved class-selective determination based on considerably lower % CV values as well as precise discrimination against other types of pesticides.

A cholinesterase (ChE)-based dipstick-type assay for the class-specific detection of organophosphate (OP) and carbamate (CM) pesticides was developed. The principle of the assay is based on inhibition of the activity of a ChE by these two families of pesticides, which is dependent on the concentration of pesticides. The proposed assay system is composed of a test strip with an acetylcholinesterase (AChE)-coated membrane and an enzyme substrate solution. The assay protocol involves incubation of the enzyme-coated strip in the pesticide-containing sample solution followed by incubation of the sample-treated strip in a chromogenic enzyme substrate solution. The color intensity is estimated by the naked eye or a reflectometer. Of the membranes tested as the enzyme support, Hybond N+ was the most suitable. Among the compounds tested as the enzyme substrate, indophenyl acetate was the best. The detectable concentration range of the dipstick assay for the OP and CM pesticides was 10(-6)-10(2) and 10(-6)-10(0) microg mL(-1), respectively. The sensitivity of the dipstick assay to the oxidized form of parathion (paraoxon) was higher than to parathion. The strip showed a large matrix effect with pesticide-spiked lettuce samples, whereas it showed a small matrix effect with pesticide-spiked rice samples.

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. [Copyright &y& Elsevier]

Presents information on a study which determined carbamate pesticides, through a flow injection-type cholinesterase biosensor. Methodology; Results and discussion; Conclusion.

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, five haptens of fenitrothion were synthesized and two of them were conjugated to proteins to be used as immunogens for production of polyclonal antibodies. Using the antibodies and a coating antigen, an indirect competitive enzyme-linked immunosorbent assay (ELISA) for fenitrothion was developed, which showed an IC50 of 3.7 ng/mL with a detection limit of 0.5 ng/mL. A direct competitive ELISA using an enzyme tracer was also developed, which showed an IC50 of 5.6 ng/mL with a detection limit of 0.4 ng/mL. The antibodies in both assays showed negligible cross-reactivity with other OP pesticides except with the insecticide parathion-methyl only in the direct ELISA. Recoveries of fenitrothion from fortified lettuce and rice samples ranged 84–116 and 100–121%, respectively. [Copyright &y& Elsevier]

In a previous study of ours, a new synthetic method for haptens of organophosphorus (OP) pesticides, with a spacer arm (aminocarboxylic acid) attached at the thiophosphate group, was developed and applied to the synthesis of haptens for the OP insecticide fenthion. Polyclonal antibodies were obtained from the haptens that were conjugated to carrier proteins. In this study, the effect of hapten heterology on immunoassay sensitivity was investigated using the antibodies and several plate-coating haptens with different types of heterology. The findings were that the degree of hapten heterology parallels the degree of sensitivity. Using the most suitable plate-coating antigen, an indirect enzyme-linked immunosorbent assay (ELISA) for fenthion was developed. The IC50 value of fenthion, under optimum conditions, was estimated to be 16 ng/ml with a detection limit of 1 ng/ml. The antibodies showed negligible cross-reactivity with other OP pesticides except with the insecticide fenitrothion. Recoveries of fenthion from fortified rice and vegetable samples ranged between 85 and 113%. [Copyright &y& Elsevier]

The synthetic method for haptens of organophosphorus (OP) pesticides with a spacer arm (amino carboxylic acid) attached at the pesticide thiophosphate group was simplified to a large extent. While the previous synthetic approach for this type of haptens requires seven steps, the present process involves only two steps. Using this process, five haptens of fenthion differing in spacer arm length (4–8 atoms) were synthesized and they were conjugated to bovine serum albumin and keyhole limpet hemocyanin to be used as immunogens. Rabbits were immunized with these hapten–protein conjugates for production of polyclonal antibodies against fenthion. The five haptens were conjugated to ovalbumin to be used as plate-coating antigens and twenty polyclonal antisera to the haptens were screened against each of the five coating antigens using noncompetitive and competitive indirect enzyme-linked immunosorbent assay (ELISA). The titer difference between the homologous and heterologous combinations was small, suggesting that heterology in spacer arm length is not important for the antigen recognition by antibodies. While the heterology in spacer length of the coating antigen had no significant effect on the sensitivity of ELISA, heterology in spacer structure of the coating antigen produced a remarkable improvement in the sensitivity of ELISA. [Copyright &y& Elsevier]

Three immunoassay systems: indirect, direct competitive enzyme-linked immunosorbent assay (IC-ELISA and DC-ELISA), fluorescence polarization immunoassay (FPIA) based on monoclonal antibodies for the detection of parathion-methyl (PM) were developed and optimized. Several PM derivatives (haptens) were conjugated to proteins and fluoresceinthiocarbamyl ethylenediamine (EDF) to obtain immunogens and competitors. The influence of immunogen and competitor structures on the assay performance was investigated. IC-ELISA was the most sensitive of all techniques developed, with a detection limit of 0.08 ng ml-1, but assay time was the longest (3.5 h per 96-well microtitre plate). DC-ELISA was easier to perform and quicker (1.5 h per 96-well microtitre plate) but less sensitive than IC-ELISA (detection limit was 0.5 ng ml-1). FPIA was the fastest and simplest (7 min per 10 samples) but the least sensitive (detection limit was 15 ng ml-1) technique. The methods were characterized by high specificity and reproducibility. The cross-reactivity for parathion-ethyl was around 30–40% for IC-ELISA and FPIA, but significantly higher (125%) for DC-ELISA. The immunoassays were applied to the analysis of PM residues in different food and environmental matrices. Methanol extracts of vegetable, fruit and soil samples were used for the analysis. Recoveries for most spiked samples averaged between 85 and 110%. The methods developed can be used for screening of food and environmental samples for PM residues without complicated clean-up. [Copyright &y& Elsevier]