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Analyse d'une protéine biomarqueur d'intoxication par des agents neurotoxiques : développement d'immunoadsorbants et de réacteurs enzymatiques, couplés en ligne à la chromatographie liquide et à la spectrométrie de masse

Abstract : The objective of this work was to develop a sample treatment method for the LC-MS2 analysis of adducts formed between organophosphorus nerve agent (OPNA) and butyrylcholinesterase, a plasmatic protein. In order to fully automate the analytical process, an immunoextraction step was coupled to an enzymatic digestion reactor (IMER), followed by microLC-MS2 analysis to detect the OPNA-peptide adduct. First, the synthesis of pepsin-based IMER was optimized. The most powerful pepsin-based IMER was applied to the digestion of HuBuChE to generate the peptide used as biomarker of OPNA intoxication that was further analyzed by nanoLC-MS2. This pepsin-based IMER was then integrated into a microLC-MS2 analytical system, allowing the on-line digestion of HuBuChE and the analysis of the target peptide. In a second step, a selective extraction method was developed by immobilizing anti-HuBuChE antibodies on a solid support, called immunosorbent. The immunoextraction protocol was developed and the immunosorbents were characterized in terms of selectivity and extraction yield before being coupled to the IMER. The feasibility of the coupling was clearly demonstrated and this automatable device was used for the analysis of HuBuChE from human plasma, spiked with OPNA. This allowed the fast and sensitive detection of OPNA-BuChE adducts in very small amounts of plasma.
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  • HAL Id : tel-01686150, version 1

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Maud Bonichon. Analyse d'une protéine biomarqueur d'intoxication par des agents neurotoxiques : développement d'immunoadsorbants et de réacteurs enzymatiques, couplés en ligne à la chromatographie liquide et à la spectrométrie de masse. Chimie-Physique [physics.chem-ph]. Université Pierre et Marie Curie - Paris VI, 2017. Français. ⟨NNT : 2017PA066228⟩. ⟨tel-01686150⟩

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