A flow immunoassay for studies of human exposure and toxicity in biological samples

This paper describes a heterogeneous competitive flow immunoassay with a high sample throughput which can be used for the screening of smaller analytes in various samples. The method is based on off-line incubation of the analyte (Ag), a fluorescent labelled tracer (Ag*) and the corresponding antibody (Ab). The separation of bound (Ab-Ag*) and free tracer (Ag*) is based on a size exclusion and reversed phase mechanism utilizing a restricted access (RA) column. The column traps the free unbound tracer (Ag*) in its hydrophobic (C18) inner cavity but excludes the large Ab-Ag* complex, which is passed on and measured by the fluorescence detector. The flow immunoassay was developed using the triazine herbicide atrazine as a model compound owing to its human toxicity and widespread use. A sample throughput of 80 samples per hour and a detection limit of 300 pg ml-1 in water were obtained. Urine samples were successfully applied for direct injections into the flow system, while for human plasma samples an additional clean-up step using solid phase extraction was efficiently included where pure extract is obtained with the highly stable and biocompatible extracting column material. The resulting detection limits for atrazine in plasma and water samples using this clean-up and trace enrichment procedure were found to be 2 ng ml-1 and 20 pg ml-1 respectively.