Development of a cell culture/ELISA assay to detect anticoagulant rodenticides and its application to analysis of rodenticide treated grain

This study describes a generic biological screening assay designed to detect anticoagulant rodenticides based on their inhibitory action on the vitamin K epoxide reductase protein complex, resulting in an accumulation of under-carboxylated prothrombin or proteins induced by vitamin K antagonism (PIVKA-II). A combined cell culture/ELISA assay was optimized to measure PIVKA-II production by the human hepatoma HepG2 cell line cultured in the presence of anticoagulant rodenticides. The specificity and sensitivity of the assay was validated using 41 grain extracts containing representative concentrations of rodenticide or appropriate nonrodenticide control compounds. In all cases, PIVKA-II produced by HepG2 cells in response to grain extracts spiked with rodenticides was detected by ELISA, while PIVKA-II was not detected in supernatants collected from cells exposed to nonrodenticide controls. This represents a novel, class-specific biological assay for the detection of anticoagulant rodenticides present in contaminated grain.