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Attempts to isolate rare antigen\u2010specific memory B cells for in\u2010depth functional analysis at the single\u2010cell level have been hindered by the lack of tools with adequate sensitivity. We applied two independent methods of protein labeling to sensitive and specific <jats:italic>ex vivo<\/jats:italic> identification of antigen\u2010specific memory B cells by flow cytometry: stringently controlled amine labeling, and sortagging, a novel method whereby a single nucleophilic fluorochrome molecule is added onto an LPETG motif carried by the target protein. We show that sortagged NadA, a major antigen in the meningococcal serogroup B vaccine, identifies NadA\u2010specific memory B cells with high sensitivity and specificity, comparable to NadA amine\u2010labeled under stringent reaction parameters in a mouse model of vaccination. We distinguish NadA\u2010specific switched MBC induced by vaccination from the background signal contributed by splenic transitional and marginal zone B cells. In conclusion, we demonstrate that protein structural data coupled with sortag technology allows the development of engineered antigens that are as sensitive and specific as conventional chemically labeled antigens in detecting rare MBC, and minimize the possibility of disrupting conformational B cell epitopes.<\/jats:p><\/jats:sec>","DOI":"10.1002\/iid3.3","type":"journal-article","created":{"date-parts":[[2013,8,2]],"date-time":"2013-08-02T09:51:23Z","timestamp":1375437083000},"page":"3-13","source":"Crossref","is-referenced-by-count":4,"title":["Optimized fluorescent labeling to identify memory B cells specific for <i>Neisseria meningitidis<\/i> serogroup B vaccine antigens <i>ex vivo<\/i>"],"prefix":"10.1002","volume":"1","author":[{"given":"Nitya","family":"Nair","sequence":"first","affiliation":[{"name":"Novartis Vaccines &amp; Diagnostics Siena Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ludovico","family":"Buti","sequence":"additional","affiliation":[{"name":"Novartis Vaccines &amp; 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