Flow injection immunotitration: extended working range for inhibition type immunodetection

Abstract

In this paper the application of a ‘merging zones’ technique common in flow injection analysis (FIA) titrations to an affinity assay for a low molecular weight ligand is presented. A pesticide immunoassay for the triazine pesticide atrazine was run either by pre-mixing a fixed amount of antibody with a given amount of sample or by sequential injection of an analyte plug followed by an antibody plug into a FIA system. The assay was performed in a binding inhibition format. Free antibodies not blocked by analyte molecules were quantified by a label-free flow-through immunosensor. In the pre-mixing approach only a single reading is obtained, which responds to changes in analyte concentration over about one order of magnitude. In the merging zones approach the mixing of the analyte and the antibody solution zone allowed to exploit a broader range of analyte and antibody concentrations. The merging zones approach showed a significantly extended working range from 0.5 μg/l to more than 20 μg/l atrazine. Potential, limitations, and possible extensions of the method like affinity titrations from a single experiment, are discussed.