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Determining an individual's humoral immune reactivity to a pathogen, autoantigen, or environmental agent is traditionally accomplished through the assessment of specific antibody levels in blood. However, in many instances, titers of specific antibodies decline over time and thus do not faithfully reveal prior antigen exposure or establishment of immunological memory. To estimate an individual's humoral immune competence, it is therefore necessary to assess functional B cell memory. Here, we describe novel B cell ELISPOT and FluoroSpot assays (collectively referred to as ImmunoSpot) that can be rapidly developed and validated to characterize the memory B cell (Bmem) repertoire specific for any desired antigen ex vivo and at single-cell resolution. Moreover, multiplexed variants of the B cell FluoroSpot assay enable high-throughput testing of antigen-specific B cells secreting distinct antibody classes and/or IgG subclasses, with minimal cell material requirements. B cell ImmunoSpot assays also enable measurement of affinity distributions within the antigen-specific Bmem compartment and permit cross-reactivity measurements that can provide insights into Bmem established against future pathogen variants. Collectively, the ImmunoSpot® system presented here is highly reproducible, and can be readily validated for regulated tests. The newly gained ability to monitor the antigen-specific Bmem compartment should catalyze a more comprehensive understanding of humoral immunity in health and disease. [ABSTRACT FROM AUTHOR]