Real-Time Cytotoxicity Assay for Rapid and Sensitive Detection of Ricin from Complex Matrices 英文参考文献.docVIP

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Real-Time Cytotoxicity Assay for Rapid and Sensitive Detection of Ricin from Complex Matrices 英文参考文献.doc

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Real-Time Cytotoxicity Assay for Rapid and Sensitive Detection of Ricin from Complex Matrices 英文参考文献

Real-TimeCytotoxicityAssayforRapidandSensitive DetectionofRicinfromComplexMatrices DianaPauly,SylviaWorbs,SebastianKirchner,OlenaShatohina,MartinB.Dorner,BrigitteG.Dorner* CenterforBiologicalSecurity-MicrobialToxins,RobertKoch-Institut,Berlin,Germany Abstract Background:Inthecontextofapotentialbioterroristattacksensitiveandfastdetectionoffunctionallyactivetoxinssuchas ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methodscurrentlyavailableforricinistheendpointcytotoxicityassay,whichsuffersfromanumberoftechnicaldeficits. Methodology/Findings:ThisworkdescribesanovelonlinecytotoxicityassayforthedetectionofactivericinandRicinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubationwithtoxinthecellstatusandthecytotoxiceffectwerevisualizedusingacharacteristiccellindex–timeprofile. For ricin, testedinconcentrations of0.06ng/mL orabove, aconcentration-dependent decreaseof cellindex correlating withcytotoxicitywasrecordedbetween3.5hand60h.Forricin,sensitivedetectionwasdeterminedafter24h,withan IC50of0.4ng/mL(foragglutinin,anIC50of30ng/mLwasobserved).Usingfunctionallyblockingantibodies,thespecificity forricinandagglutininwasshown.Fordetectionfromcomplexmatrices,ricinwasspikedintoseveralfoodmatrices,andan IC50rangingfrom5.6to200ng/mLwasobserved.Additionally,theassayprovedtobeusefulindetectingactivericinin environmentalsamplematerials,asshownfororganicfertilizercontainingR.communismaterial. Conclusions/Significance: The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficientlythanpreviousapproachesbasedonendpointmeasurement.Moreimportantly,thereal-timecytotoxicityassay