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 between an unmodified multivalent lectin and fluorochrome-labeled dextrans bearing specific sugar ligands (analyte-analog). The measuring principle relies on the fact that one portion of the dextran is coupled with an emitter dye fluorescein isothiocyanate (FITC), and the other one with an acceptor dye (isothiocyanate-derivatives of rhodamine). In absence 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 (~5 nm) so that fluorescence resonance energy transfer (FRET) occurs between fluorescein and rhodamine. Hence the fluorescence is quenched. The displacement of dextrans by a specific sugar results in the dissociation of the complex and in an inverse increase in fluorescence which is proportional to the sugar concentration. The paper describes experiments proofing the conceptual idea of this fluorescence assay on two examples: a glucose and galactose-specific assay system. The glucosespecific assay consisted of Concanavalin A (Con A) and fluorescein and rhodamine-labeled dextran (Mr 2000 kDa) grafted with mannose. The galactose-specific assay was composed of Ricinus communis agglutinin (RCAI) and fluorescein and rhodamine-labeled dextran (Mr 2000 kDa) grafted with lactose. The reversibility and response time of both assays inside a single dialysis 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 sensor showed a good measurable fluorescence signal over a period of 11 days. The use of this assay for antibody/antigen system is proposed.