{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,9,29]],"date-time":"2025-09-29T15:49:46Z","timestamp":1759160986018,"version":"3.37.3"},"reference-count":45,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2022,9,1]],"date-time":"2022-09-01T00:00:00Z","timestamp":1661990400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100006492","name":"Division of Intramural Research, National Institute of Allergy and Infectious Diseases","doi-asserted-by":"publisher","award":["R01AI104579"],"award-info":[{"award-number":["R01AI104579"]}],"id":[{"id":"10.13039\/100006492","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS ONE"],"abstract":"<jats:p>Generating specific monoclonal antibodies (mAbs) that neutralize multiple antigen variants is challenging. Here, we present a strategy to generate mAbs that bind seven subtypes of botulinum neurotoxin serotype F (BoNT\/F) that differ from each other in amino acid sequence by up to 36%. Previously, we identified 28H4, a mouse mAb with poor cross-reactivity to BoNT\/F1, F3, F4, and F6 and with no detectable binding to BoNT\/F2, F5, or F7. Using multicolor labeling of the different BoNT\/F subtypes and fluorescence-activated cell sorting (FACS) of yeast displayed single-chain Fv (scFv) mutant libraries, 28H4 was evolved to a humanized mAb hu6F15.4 that bound each of seven BoNT\/F subtypes with high affinity (K<jats:sub>D<\/jats:sub> 5.81 pM to 659.78 pM). In contrast, using single antigen FACS sorting, affinity was increased to the subtype used for sorting but with a decrease in affinity for other subtypes. None of the mAb variants showed any binding to other BoNT serotypes or to HEK293 or CHO cell lysates by flow cytometry, thus demonstrating stringent BoNT\/F specificity. Multicolor FACS-mediated antibody library screening is thus proposed as a general method to generate multi-specific antibodies to protein subtypes such as toxins or species variants.<\/jats:p>","DOI":"10.1371\/journal.pone.0273512","type":"journal-article","created":{"date-parts":[[2022,9,1]],"date-time":"2022-09-01T17:57:10Z","timestamp":1662055030000},"page":"e0273512","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":5,"title":["Multicolor fluorescence activated cell sorting to generate humanized monoclonal antibody binding seven subtypes of BoNT\/F"],"prefix":"10.1371","volume":"17","author":[{"given":"Yongfeng","family":"Fan","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhengda","family":"Sun","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fraser","family":"Conrad","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weihua","family":"Wen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lequn","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jianlong","family":"Lou","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yu","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shauna","family":"Farr-Jones","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0562-3517","authenticated-orcid":true,"given":"James 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Simmons","year":"2007","journal-title":"PLoS Med"},{"issue":"7870","key":"pone.0273512.ref006","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1038\/s41586-021-03720-y","article-title":"In vivo monoclonal antibody efficacy against SARS-CoV-2 variant strains","volume":"596","author":"RE Chen","year":"2021","journal-title":"Nature"},{"key":"pone.0273512.ref007","doi-asserted-by":"crossref","first-page":"86","DOI":"10.1128\/mr.46.1.86-94.1982","article-title":"Bacterial toxins: a table of lethal amounts","volume":"46","author":"MD Gill","year":"1982","journal-title":"Microbiol Rev"},{"key":"pone.0273512.ref008","doi-asserted-by":"crossref","first-page":"1091","DOI":"10.1006\/jmbi.1999.2945","article-title":"Sequence homology and structural analysis of the Clostridial neurotxins","volume":"291","author":"DB Lacy","year":"1999","journal-title":"J Mol 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activity","volume":"10","author":"Y Fan","year":"2015","journal-title":"PLoS One"},{"key":"pone.0273512.ref036","doi-asserted-by":"crossref","first-page":"W296","DOI":"10.1093\/nar\/gky427","article-title":"SWISS-MODEL: homology modelling of protein structures and complexes","volume":"46","author":"A. Waterhouse","year":"2018","journal-title":"Nucleic Acids Res"},{"issue":"1","key":"pone.0273512.ref037","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1007\/BF02102452","article-title":"Molecular anatomy: phyletic relationships derived from three-dimensional structures of proteins","volume":"30","author":"MS Johnson","year":"1990","journal-title":"J Mol Evol"},{"issue":"Pt 12 Pt 1","key":"pone.0273512.ref038","doi-asserted-by":"crossref","first-page":"2256","DOI":"10.1107\/S0907444904026460","article-title":"Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions","volume":"60","author":"E Krissinel","year":"2004","journal-title":"Acta Crystallogr D Biol Crystallogr"},{"issue":"13","key":"pone.0273512.ref039","doi-asserted-by":"crossref","first-page":"1605","DOI":"10.1002\/jcc.20084","article-title":"UCSF Chimera\u2014a visualization system for exploratory research and analysis","volume":"25","author":"EF Pettersen","year":"2004","journal-title":"J Comput Chem"},{"issue":"8","key":"pone.0273512.ref040","doi-asserted-by":"crossref","first-page":"1059","DOI":"10.1001\/jama.285.8.1059","article-title":"Botulinum toxin as a biological weapon: medical and public health management","volume":"285","author":"SS Arnon","year":"2001","journal-title":"JAMA: the journal of the American Medical Association"},{"issue":"4","key":"pone.0273512.ref041","doi-asserted-by":"crossref","first-page":"265","DOI":"10.4103\/1735-5362.212043","article-title":"Immunization of mice by a multimeric L2-based linear epitope (17\u201336) from HPV type 16\/18 induced cross reactive neutralizing antibodies","volume":"12","author":"FM Khiavi","year":"2017","journal-title":"Res Pharm Sci"},{"issue":"11","key":"pone.0273512.ref042","doi-asserted-by":"crossref","first-page":"e1000197","DOI":"10.1371\/journal.ppat.1000197","article-title":"Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway","volume":"4","author":"J Sui","year":"2008","journal-title":"PLoS Pathog"},{"issue":"6359","key":"pone.0273512.ref043","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1126\/science.aan8630","article-title":"Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques","volume":"358","author":"L Xu","year":"2017","journal-title":"Science"},{"issue":"8","key":"pone.0273512.ref044","doi-asserted-by":"crossref","first-page":"648","DOI":"10.1038\/nri1416","article-title":"Seventeen-colour flow cytometry: unravelling the immune system","volume":"4","author":"SP Perfetto","year":"2004","journal-title":"Nature Rev Immunol"},{"issue":"6","key":"pone.0273512.ref045","doi-asserted-by":"crossref","first-page":"3841","DOI":"10.4049\/jimmunol.179.6.3841","article-title":"Kinetic, affinity, and diversity limits of human polyclonal antibody responses against tetanus toxoid","volume":"179","author":"TR Poulsen","year":"2007","journal-title":"J Immunol"}],"container-title":["PLOS ONE"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0273512","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,9,1]],"date-time":"2022-09-01T17:58:06Z","timestamp":1662055086000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0273512"}},"subtitle":[],"editor":[{"given":"Kevin A.","family":"Henry","sequence":"first","affiliation":[],"role":[{"role":"editor","vocabulary":"crossref"}]}],"short-title":[],"issued":{"date-parts":[[2022,9,1]]},"references-count":45,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2022,9,1]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0273512","relation":{},"ISSN":["1932-6203"],"issn-type":[{"type":"electronic","value":"1932-6203"}],"subject":[],"published":{"date-parts":[[2022,9,1]]}}}