Multiplexed ImmunoSpot™ enables streamlined assessment of antigen-specific B cells; lessons toward maximizing cell utilization and increasing sample throughput

There are few techniques capable of detecting rare antigen-specific B cells while also providing information regarding their abundance, Ig class/subclass usage and functional affinity. The ImmunoSpot™ assay approach is suited for such antigen-specific B cell assessments since it provides information at single-cell resolution for individual ASC. Moreover, recently introduced innovations in the implementation of this assay modality have opened the door to testing larger sample sets. Towards this objective, multiplexed B cell FluoroSpot assays reduce the cell material requirement through parallel assessment of multiple Ig classes or IgG subclasses. Nevertheless, accurate determination of ASC frequencies, especially in the context of diverse antigen panels and complex exposure histories, is best achieved through evaluating multiple cell inputs to accommodate the inherent variability present within and amongst individual donors. The scope of this research effort was to further optimize the assay pipeline to facilitate high-throughput suitable assessment of antigen-specific ASC. This goal has been achieved through introducing advancements in the image analysis software component, improving both the time requirement and precision of downstream data analysis. Collectively, implementation of these assay optimizations serves to increase the feasibility of making high-throughput assessment of antigen-specific ASC using the ImmunoSpot™ approach a routine practice in immune monitoring.