Paraquat enzyme-immunoassays in biological samples: assessment of the effects of hapten–protein bridge structures on assay sensitivity

Previously unreported paraquat derivatives were prepared and used to develop enzyme-immunoassay methods for paraquat in serum and urine matrices. The study involved comparison of the effects of novel paraquat derivatives made of methyl and ethyl-4,4′-bipyridinium and cyanuric chloride (heterologous bridges) or valeric acid (homologous bridges) on the ability of paraquat standards to inhibit binding of the antibody to adsorbed hapten–protein plate coating antigens prepared by coupling the derivatives to gelatine. The comparison showed striking differences in assay sensitivity due to the hapten bridge binding phenomenon where the heterologous bridge conjugates enabled achievement of sensitivity levels several orders of magnitude greater than the homologous structures. The constructed ELISA showed minimal detection limit in the range 4 pg mL−1 in the buffer systems and less then 100 pg mL−1 in charcoal-stripped human and horse sera and human urine. The study presents details of synthesis of novel paraquat derivatives and a highly sensitive ELISA. In addition the investigation demonstrates the critical importance of judicious selection of hapten-bridge structures to achieve improved levels of detection limits of paraquat immunoassays. The reported assay is suitable for use in monitoring of paraquat levels in exposed persons or animals and for emergency diagnostic tests.