Monia Bardelli, Liliana Alleri, Francesca Angiolini, Francesca Buricchi, Simona Tavarini, Chiara Sammicheli, Sandra Nuti, Elena Degl'Innocenti, Isabelle Isnardi, Elena Fragapane, Giuseppe Del Giudice, Flora Castellino, Grazia Galli
Understanding the impact that human memory B-cells (MBC), primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA) is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube...
2013: PloS One