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To test the ability of the technique to provide insights into the functional properties of monoclonal antibody (mAb) epitopes, we used here a well-characterized epitope of meningococcal factor H binding protein (fHbp), which is recognized by mAb 12C1. An fHbp library, engineered on a lambda phage vector enabling surface expression of polypeptides of widely different length, was subjected to massive parallel sequencing of the phage inserts after affinity selection with the 12C1 mAb. We detected dozens of unique antibody-selected sequences, the most enriched of which (designated as FrC) could largely recapitulate the ability of fHbp to bind mAb 12C1. Computational analysis of the cumulative enrichment of single amino acids in the antibody-selected fragments identified two overrepresented stretches of residues (H248-K254 and S140-G154), whose presence was subsequently found to be required for binding of FrC to mAb 12C1. Collectively, these results suggest that the PROFILER technology can rapidly and reliably identify, in the context of complex conformational epitopes, discrete \u201chot spots\u201d with a crucial role in antigen-antibody interactions, thereby providing useful clues for the functional characterization of the epitope.<\/jats:p>","DOI":"10.1038\/srep31458","type":"journal-article","created":{"date-parts":[[2016,8,17]],"date-time":"2016-08-17T09:07:24Z","timestamp":1471424844000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Functional characterization of a monoclonal antibody epitope using a lambda phage display-deep sequencing platform"],"prefix":"10.1038","volume":"6","author":[{"given":"Maria","family":"Domina","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Veronica","family":"Lanza Cariccio","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Salvatore","family":"Benfatto","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mario","family":"Venza","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabella","family":"Venza","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erica","family":"Borgogni","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Flora","family":"Castellino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Angelina","family":"Midiri","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Roberta","family":"Galbo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Letizia","family":"Romeo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carmelo","family":"Biondo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vega","family":"Masignani","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giuseppe","family":"Teti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Franco","family":"Felici","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Concetta","family":"Beninati","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2016,8,17]]},"reference":[{"key":"BFsrep31458_CR1","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1038\/sj.icb.7100004","volume":"85","author":"YH Zhou","year":"2007","unstructured":"Zhou, Y. H., Chen, Z., Purcell, R. H. & Emerson, S. U. Positive reactions on Western blots do not necessarily indicate the epitopes on antigens are continuous. Immunology and cell biology 85, 73\u201378, doi: 10.1038\/sj.icb.7100004 (2007).","journal-title":"Immunology and cell biology"},{"key":"BFsrep31458_CR2","doi-asserted-by":"publisher","first-page":"185","DOI":"10.1002\/jms.1280","volume":"43","author":"T Pimenova","year":"2008","unstructured":"Pimenova, T. et al. Epitope mapping on bovine prion protein using chemical cross-linking and mass spectrometry. Journal of mass spectrometry: JMS 43, 185\u2013195, doi: 10.1002\/jms.1280 (2008).","journal-title":"Journal of mass spectrometry: JMS"},{"key":"BFsrep31458_CR3","doi-asserted-by":"publisher","first-page":"530","DOI":"10.1016\/j.jsb.2010.10.014","volume":"173","author":"J Rappsilber","year":"2011","unstructured":"Rappsilber, J. The beginning of a beautiful friendship: cross-linking\/mass spectrometry and modelling of proteins and multi-protein complexes. Journal of structural biology 173, 530\u2013540, doi: 10.1016\/j.jsb.2010.10.014 (2011).","journal-title":"Journal of structural biology"},{"key":"BFsrep31458_CR4","doi-asserted-by":"publisher","first-page":"747","DOI":"10.1038\/322747a0","volume":"322","author":"DJ Barlow","year":"1986","unstructured":"Barlow, D. J., Edwards, M. S. & Thornton, J. M. Continuous and discontinuous protein antigenic determinants. Nature 322, 747\u2013748, doi: 10.1038\/322747a0 (1986).","journal-title":"Nature"},{"key":"BFsrep31458_CR5","doi-asserted-by":"publisher","first-page":"706","DOI":"10.1038\/srep00706","volume":"2","author":"EP Hudson","year":"2012","unstructured":"Hudson, E. P., Uhlen, M. & Rockberg, J. Multiplex epitope mapping using bacterial surface display reveals both linear and conformational epitopes. 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Be. and G.T. hold shares in Scylla Biotech S.r.l., which has submitted a patent application for the PROFILER technology. E.B and V.M. are full-time employees of GSK Vaccines.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"31458"}}