Optimized fluorescent labeling to identify memory B cells specific for Neisseria meningitidis serogroup B vaccine antigens ex vivo (pages 3–13)
Nitya Nair, Ludovico Buti, Elisa Faenzi, Francesca Buricchi, Sandra Nuti, Chiara Sammicheli, Simona Tavarini, Maximilian W.L. Popp, Hidde Ploegh, Francesco Berti, Mariagrazia Pizza, Flora Castellino, Oretta Finco, Rino Rappuoli, Giuseppe Del Giudice, Grazia Galli and Monia Bardelli
Article first published online: 1 AUG 2013 | DOI: 10.1002/iid3.3
We describe two independent methods to optimize protein antigen labeling with fluorochromes for FACS identification and analysis of rare antigen-specific memory B cells: conventional amine labeling with stringently controlled reaction parameters, and a novel site-specific method, named sortagging, where nucleophilic fluorochromes are added in known numbers to LPTEG motifs expressed on the target proteins by using the staphylococcal sortase A. Using the meningococcal serogroup B NadA antigen and a mouse model of vaccination, we show that both methods permit the specific identification of all NadA-specific memory B cells by FACS directly in ex vivo samples. We demonstrate that both techniques minimize the degree of labeling while maintaining adequate signal to noise ratios for sensitive and identification of rare memory B cells specific for at least two different antigens. This method facilitates accurate control of antigen integrity thus providing the potential for more valid analysis and sorting of antigen-specific B cell repertoires and a powerful tool for best understanding B cell dynamics following vaccination or infection.