Multiplex analysis of antigen-specific memory B cells in humans using reversed B-cell FluoroSpot

Analysis of B-cell specificities at the single cell level provides important information on how the B-cell compartment responds when challenged by infection or vaccination. We recently developed a reversed B-cell FluoroSpot assay and showed that it could be used to detect B cells specific for different antigens simultaneously in a mouse model. The aim of this study was to further develop the method to detect and quantify antigen-specific memory B cells (MBCs) in humans where circulating antigen-specific cells are less frequent. We show that MBCs specific for three antigens, tetanus toxoid, hepatitis B surface antigen and cytomegalovirus pp65, could be detected simultaneously in one well. In addition to enumerating antigen-specific MBCs, we also assessed the spot volume to estimate the intensity of the response in individual cells and found this to be a new and sensitive approach to study MBC responses after vaccination. This unique B-cell FluoroSpot approach provides a simple and sensitive multiplex analysis of MBCs and can be adapted to most antigens and host species. •A multiplex B-cell FluoroSpot assay was developed for studies on human memory B cells.•Antigens from hepatitis B, cytomegalovirus and tetanus toxoid were expressed recombinantly with different tags.•Memory B cells against antigens from hepatitis B, cytomegalovirus and tetanus were detected simultaneously.•Antigen-specific memory B cells were measured before and after hepatitis B vaccination.•Data on spot intensity and size provided from the FluoroSpot reader offers a more detailed analysis compared to only the spot frequency.