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 (5-trifluoromethyl-2-pyridyloxy)phenoxy)-propionic acid (fu silade), was derivatized with the fluorescent probe 4-bromomethyl-7-me thoxycoumarin (BrMmC). The labelled hapten (fusilade-coumarin, F-C) wa s incubated with anti-fusilade IgG and the fluorescence spectra, inten sity and time resolved fluorescence decay of the coumarin derivative w ere measured to study the complexation process of the hapten-antibody system. The fluorescence decay was fit to a two component decay. The m agnitude and proportion of these components remained constant but a th ree-fold increase in intensity was observed during the complexation pr ocess. No shifts in the wavelength of the maximum emission occurred du ring the complexation. The mechanism of the fluorescence intensity res ponse was studied, and it was determined that the increase in intensit y observed during the direct fluorescence assay was not due to changes of mobility, polarity or viscosity in the vicinity of the probe. A fu ll examination of fluorescence quenching processes indicated formation of non-emissive ground state complexes (dimers or higher order aggreg ates) at probe concentrations above 10(-6) M. A homogeneous competitiv e binding assay indicated that the system did not demonstrate competit ive binding as the addition of non-fluorescent fusilade did not result in a displacement of labelled fusilade and thus a decrease in intensi ty. An assay involving mixing of varying amounts of unlabelled fusilad e with a known concentration of F-C in the presence of anti-fusilade I gG was determined to be feasible.