Fluorescent Labeling of Antibody Fragments Using Split GFP 英文参考文献.docVIP

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Fluorescent Labeling of Antibody Fragments Using Split GFP 英文参考文献.doc

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Fluorescent Labeling of Antibody Fragments Using Split GFP 英文参考文献

FluorescentLabelingofAntibodyFragmentsUsingSplit GFP FortunatoFerrara,PawelListwan,GeoffreyS.Waldo,AndrewR.M.Bradbury* BioscienceDivision,LosAlamosNationalLaboratory,LosAlamos,NewMexico,UnitedStatesofAmerica Abstract Antibody fragments are easily isolated from in vitro selection systems, such as phage and yeast display. Lacking the Fc portion of the antibody, they are usually labeled using small peptide tags recognized by antibodies. In this paper we presentanefficientmethodtofluorescentlylabelsinglechainFvs(scFvs)usingthesplitgreenfluorescentprotein(GFP) system.A13aminoacidtag,derivedfromthelastbetastrandofGFP(termedGFP11),isfusedtotheCterminusofthescFv. Thistaghasbeenengineeredtobenon-perturbing,andwewereabletoshowthatitexertednoeffectonscFvexpression orfunctionalitywhencomparedtoascFvwithouttheGFP11tag.Effectivefunctionalfluorescentlabelingisdemonstrated in a number of different assays, including fluorescence linked immunosorbant assays, flow cytometry and yeast display. Furthermore,wewereabletoshowthatthissplitGFPsystemcanbeusedtodeterminetheconcentrationofscFvincrude samples,aswellanestimateofantibodyaffinity,withouttheneedforantibodypurification.Weanticipatethissystemwill beofwidespreadinterestinantibodyengineeringandinvitrodisplaysystems. Citation:FerraraF,ListwanP,WaldoGS,BradburyARM(2011)FluorescentLabelingofAntibodyFragmentsUsingSplitGFP.PLoSONE6(10):e25727.doi:10.1371/ journal.pone.0025727 Editor:DafyddJones,CardiffUniversity,UnitedKingdom ReceivedMarch24,2011;AcceptedSeptember9,2011;PublishedOctober5,2011 Copyright: ? 2011 Ferrara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding:FundedbytheNIH(P50GM085273andR01-HG004852-01A1).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish, orpreparationofthemanuscript. CompetingInterests:Theauthorshavedecl