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dc.contributor.authorMankowska, Sylwiaen
dc.contributor.authorGatti-Lafranconi, Pietroen
dc.contributor.authorChodorge, Men
dc.contributor.authorSridharan, Sen
dc.contributor.authorMinter, RRen
dc.contributor.authorHollfelder, Florianen
dc.date.accessioned2016-12-19T20:40:30Z
dc.date.available2016-12-19T20:40:30Z
dc.date.issued2016-11-07en
dc.identifier.issn2045-2322
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/261655
dc.description.abstractAffinity panning of large libraries is a powerful tool to identify protein binders. However, panning rounds are followed by the tedious re-screening of the clones obtained to evaluate binders precisely. In a first application of Bead Surface Display (BeSD) we show successful $\textit{in vitro}$ affinity selections based on flow cytometric analysis that allows fine quantitative discrimination between binders. Subsequent consensus analysis of the resulting sequences enables identification of clones that bind tighter than those arising directly from the experimental selection output. This is demonstrated by evolution of an anti-Fas receptor single-chain variable fragment (scFv) that was improved 98-fold $\textit{vs}$ the parental clone. Four rounds of quantitative screening by fluorescence-activated cell sorting of an error-prone library based on fine discrimination between binders in BeSD were followed by analysis of 200 full-length output sequences that suggested a new consensus design with a $K_d$ ~ 140pM. This approach shortens the time and effort to obtain high affinity reagents and its cell-free nature transcends limitations inherent in previous $\textit{in vivo}$ display systems.
dc.description.sponsorshipS.A.M. was supported by a CASE studentship from the Biological and Biotechnological Sciences Research Council and MedImmune. F.H. is an Investigator of the European Research Council.
dc.languageengen
dc.language.isoenen
dc.publisherNature Publishing Group
dc.rightsAttribution 4.0 Internationalen
dc.rightsAttribution 4.0 Internationalen
dc.rightsAttribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectantibody therapyen
dc.subjectbiotechnologyen
dc.subjecthigh-throughput screeningen
dc.subjectmolecular engineeringen
dc.titleA Shorter Route to Antibody Binders $\textit{via}$ Quantitative $\textit{in vitro}$ Bead-Display Screening and Consensus Analysisen
dc.typeArticle
prism.number36391en
prism.publicationDate2016en
prism.publicationNameScientific Reportsen
prism.volume6en
dc.identifier.doi10.17863/CAM.6863
dcterms.dateAccepted2016-10-11en
rioxxterms.versionofrecord10.1038/srep36391en
rioxxterms.versionVoRen
rioxxterms.licenseref.urihttp://creativecommons.org/licenses/by/4.0/en
rioxxterms.licenseref.startdate2016-11-07en
dc.contributor.orcidHollfelder, Florian [0000-0002-1367-6312]
dc.identifier.eissn2045-2322
rioxxterms.typeJournal Article/Reviewen
pubs.funder-project-idBBSRC (C19399)
cam.issuedOnline2016-11-07en
cam.orpheus.successThu Jan 30 12:56:50 GMT 2020 - The item has an open VoR version.*
rioxxterms.freetoread.startdate2100-01-01


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International