title: Improvement of acetylcholinesterase-based assay for organophosphates in way of identification by reactivators.
abstract: Organophosphates present serious fulmination in several aspects of human life. Detection of organophosphates is frequently based on following acetylcholinesterase (AChE) inhibition. Although limit of detection and sensitivity for AChE-based assays seem to be intriguing, the identification of organophosphates is not currently efficient in this way. We introduce an improvement of AChE-based assay by reactivators using a selective come-back of AChE activity after previous inhibition. We have chosen four organophosphates: paraoxon-ethyl, paraoxon-methyl, trichlorfon, methamidophos as representative pesticides and the three most available reactivators: HI-6, obidoxime, pralidoxime. Reactivation was realized in the 96-wells photometric microplates and activity of human recombinant AChE was followed by reaction of Ellman's reagent with one of enzyme digestion product: thiocholine. Distinguishing of reactivation efficacy was judged by the independent two population t-test. The most significant identification was based on methamidophos inhibited AChE reactivation by HI-6 or pralidoxime and paraoxon-ethyl inhibited AChE by obidoxime; moreover, identification of trichlorfon and paraoxon-methyl was possible, too. Practical impact of described method is discussed.
authors.affiliation: Center of Advanced Studies, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic. rau@atlas.cz
authors.forename: Miroslav
authors.initials: M
authors.lastname: Pohanka
authors.forename: Daniel
authors.initials: D
authors.lastname: Jun
authors.forename: Kamil
authors.initials: K
authors.lastname: Kuca
mesh_terms: D000110:Acetylcholinesterase / Q000032:analysis* / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D010755:Organophosphates / Q000032:analysis* / Q000378:metabolism*
chemical_list: D010755:Organophosphates; D000110:Acetylcholinesterase