Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo
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Nair-2013-Optimized fluorescen.pdf
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Author(s)
Nair, Nitya
Buti, Ludovico
Faenzi, Elisa
Buricchi, Francesca
Nuti, Sandra
Sammicheli, Chiara
Tavarini, Simona
Popp, Maximilian W.L.
Ploegh, Hidde
Berti, Francesco
Date Issued
October 2013
Journal
Immunity, Inflammation and Disease
Publisher
Wiley Blackwell
Citation
Nair, Nitya, Ludovico Buti, Elisa Faenzi, Francesca Buricchi, Sandra Nuti, Chiara Sammicheli, Simona Tavarini, et al. “ Optimized Fluorescent Labeling to Identify Memory B Cells Specific for Neisseria Meningitidis Serogroup B Vaccine Antigens Ex Vivo .” Immun. Inflamm. Dis. 1, no. 1 (October 2013): 3–13.
Version
Final published version
Abstract
Antigen-specific memory B cells generate anamnestic responses and high affinity antibodies upon re-exposure to pathogens. Attempts to isolate rare antigen-specific memory B cells for in-depth functional analysis at the single-cell level have been hindered by the lack of tools with adequate sensitivity. We applied two independent methods of protein labeling to sensitive and specific ex vivo identification of antigen-specific 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-specific memory B cells with high sensitivity and specificity, comparable to NadA amine-labeled under stringent reaction parameters in a mouse model of vaccination. We distinguish NadA-specific 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.
MIT Department
Massachusetts Institute of Technology. Department of Biology
Whitehead Institute for Biomedical Research
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DOI of Published Version
https://doi.org/10.1002/iid3.3
