COMPETITIVE-BINDING ASSAY-METHOD BASED ON FLUORESCENCE QUENCHING OF LIGANDS HELD IN CLOSE PROXIMITY BY A MULTIVALENT RECEPTOR

A variant of a fluorescence quenching affinity assay is described that is based on intermolecular complexation due to specific interaction b etween an unmodified multivalent lectin and fluorochrome-labeled dextr ans bearing specific sugar ligands (analyte-analog). The measuring pri nciple relies on the fact that one portion of the dextran is coupled w ith an emitter dye fluorescein isothiocyanate (FITC), and the other on e with an acceptor dye (isothiocyanate-derivatives of rhodamine). In a bsence of a specific sugar, the bridging of rhodamine and fluorescein- labeled dextrans by the lectin results in the formation of a sandwich- like fluorescein-dextran/lectin/rhodamine-dextran complex in which the two forms of dextran are very close together (similar to 5 nm) so tha t fluorescence resonance energy transfer (FRET) occurs between fluores cein and rhodamine. Hence the fluorescence is quenched. The displaceme nt of dextrans by a specific sugar results in the dissociation of the complex and in an inverse increase in fluorescence which is proportion al to the sugar concentration. The paper describes experiments proofin g the conceptual idea of this fluorescence assay on two examples: a gl ucose and galactose-specific assay system. The glucose-specific assay consisted of Concanavalin A (Con A) and fluorescein and rhodamine-labe led dextran (M-r 2000 kDa) grafted with mannose. The galactose-specifi c assay was composed of Ricinus communis agglutinin (RCAI) and fluores cein and rhodamine-labeled dextran (M-r 2000 kDa) grafted with lactose . The reversibility and response time of both assays inside a single d ialysis hollow fiber, which was fixed within a flow through cell of a fluorometer, were studied during changes of the sugar concentrations. The response time of the sensor fiber was about 4-5 min. The glucose s ensor showed a good measurable fluorescence signal over a period of 11 days. The use of this assay for antibody/antigen system is proposed.