Towards a homogeneous fluorescence assay for a herbicide: characterization of the interactions of fusilade, bromomethylmethoxy coumarin derivatized fusilade, and anti-fusilade IgG antibody

The hapten 2-(4-(5-trifluoromethyl-2-pyridyloxy)phenoxy)-propionic acid (fusilade), was derivatized with the fluorescent probe 4-bromomethyl-7-methoxycoumarin (BrMmC). The labelled hapten (fusilade-coumarin, F-C) was incubated with antifusilade IgG and the fluorescence spectra, intensity and time resolved fluorescence decay of the coumarin derivative were measured to study the complexation process of the hapten-antibody system. The fluorescence decay was fit to a two component decay. The magnitude and proportion of these components remained constant but a three-fold increase in intensity was observed during the complexation process. No shifts in the wavelength of the maximum emission occurred during the complexation. The mechanism of the fluorescence intensity response was studied, and it was determined that the increase in intensity observed during the direct fluorescence assay was not due to changes of mobility, polarity or viscosity in the vicinity of the probe. A full examination of fluorescence quenching processes indicated formation of non-emissive ground state complexes (dimers or higher order aggregates) at probe concentrations above 10−6M. A homogeneous competitive binding assay indicated that the system did not demonstrate competitive binding as the addition of non-fluorescent fusilade did not result in a displacement of labelled fusilade and thus a decrease in intensity. An assay involving mixing of varying amounts of unlabelled fusilade with a known concentration of F-C in the presence of anti-fusilade IgG was determined to be feasible.