Solid-Phase Immobilized Tripod for Fluorescent Renewable Immunoassay. A Concept for Continuous Monitoring of an Immunoassay Including a Regeneration of the Solid Phase

Abstract

A new concept of immunoassay based on the use of a trifunctional reagent (tripod) and fluorescence resonance energy transfer (FRET) phenomenon is described. This procedure involves differential steps: (1) the tripod bearing (i) a fluorophore, (ii) a molecule structurally close to the target, and (iii) a linker reacts with the solid phase; (2) the solid phase is further activated with an anti-target antibody labeled with a quencher molecule, generating the decrease of the fluorophore emission via FRET; (3) FRET being distance dependent, the presence of the target by competing with the tripod for binding the quencher-labeled antibody leads to a rise of the fluorescence signal; (4) the solid phase is reactivated simply, by adding the quencher-labeled antibody. This method was evaluated in microtiter plates using the susbtance P as model while fluorescein and TAMRA were used as donor and acceptor, respectively. Results clearly illustrated the interest of the method, by allowing (i) a simple regeneration procedure, without requiring any drastic treatment, (ii) a direct fluorescence measurement onto the solid support, leading to a localized and cumulative signal, (iii) an increase of the signal when detecting the target, unlike classical competitive immunoassays, and (iv) a real-time monitoring of the competition and regeneration steps.