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Design of fluorescence immunoassays. Perspectives for continuous monitoring of biological warface agents.

Abstract : This thesis was devoted to the design of fluorescence immunoassays aiming at the detection of biological warfare agents. We developed a new concept of competitive immunoassay for continuous flow detection involving a fluorescence resonance energy transfer (FRET). This procedure is based on the use of an heterotrifunctional reagent (tripod) bearing i) a fluorophore donor (D), ii) a molecule structurally close to the target and iii) a linker to the solid phase. The binding of an anti-target antibody labeled with an acceptor molecule (A) on the tripod generates the decrease of the donor emission via the FRET phenomenon (quenching). The presence of the target competiting with the tripod for the binding to the antibody leads to an increase of the fluorescence signal. The solid phase can be easily further reactivated by adding the labeled antibody. This method was evaluated in microtiter plates using small molecules (substance P, atrazine, aflatoxine) and larger proteins (β-lactoglobuline, botulinum toxin, ricin). Spectral properties of the dyes, A/D ratio and A-D distance were demonstrated to have largely influenced FRET efficiency and hereby the sensitivity of the assay. The sensitivity also strongly depends on the binding characteristics of the antibody. The main difficulty lies on the relative affinity characteristics of the antibody towards the tripod and the analyte, since the antibody should exhibit ideally a strong affinity for the tripod and even stronger affinity for the analyte. Assays performed in glass microcells using a CCD camera and a fluorescence microscope allowed continuous-flow detection and are promising for the build-up of a biosensor. We also present a simple method for performing rapid assays in microtiter plates thanks to the difference in fluorescence signal observed between free and immobilized dyes which proved to be efficient for the design of a simultaneous multiplexed immunoassay.
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Submitted on : Friday, February 20, 2009 - 8:00:00 AM
Last modification on : Friday, February 20, 2009 - 8:00:00 AM
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Laure-Marie Neuburger. Design of fluorescence immunoassays. Perspectives for continuous monitoring of biological warface agents.. Chemical Sciences. AgroParisTech, 2006. English. ⟨NNT : 2006INAP0042⟩. ⟨pastel-00004770⟩

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