Sensitive detection of triazine and phenylurea pesticides in pure organic solvent by enzyme linked immunosorbent assay (ELISA): stabilities, solubilities and sensitivities Original Research Article

Triazine and phenylurea pesticides could be detected in pure ethanol and hexane by enzyme linked immunosorbent assay (ELISA), using microtiter plates for covalent immobilization of the respective monoclonal antibodies K4E7 and B76-BF5. Polystyrene microtiter plates were found to be resistant against several organic solvents. From those, hexane and ethanol were chosen, as they are common extraction solvents for many pesticides. For ethanol, dependence of solvent concentration on antibody and marker enzyme stability and antibody affinity was investigated. The thermal stability had a minimum at 50–80% ethanol. At low temperatures, stabilities were sufficiently high for ELISA experiments in the whole ethanol concentration range from 0 to 99% v/v. The test midpoints IC50 of the immunoassays were at least 30-fold higher, when the antigen was presented in ethanol, compared with pure water. However, a lower test midpoint was observed when the phenylurea antigen was dissolved in hexane instead of water. Detection limits below 0.2 ng ml−1 were obtained for both atrazine and phenylurea OC-2 in hexane. The sensitivities and cross-reactivities in the three solvents water, ethanol and hexane could be correlated with the solubilities of the antigens. For example, the phenylurea antigens were less soluble in hexane than in water, and therefore, their binding to the antibody was improved in hexane. These results can be explained by the hydrophobic effect. The method is potentially applicable to hydrophobic pesticides, which have to be extracted with a hydrophobic solvent, e.g. hexane.