Extending the Detection Limit of Rare Antigen-Specific B Cells in ImmunoSpot® to One in a Million—And Possibly Lower

Despite clonal expansion during a primary immune response, or after sub-sequent antigen encounters, the frequency of memory B cells (Bmem) specific for an antigen remains low, making their detection difficult. However, unlike serum an-tibodies, which have a short half-life in vivo and thus require continuous replen-ishment to maintain stable titers, circulating Bmem are long-lived; they preserve immunological preparedness through their ability to rapidly engage in recall re-sponses and differentiate into antibody-secreting cells (ASCs) upon antigen en-counter. Consequently, developing means for the reliable detection of anti-gen-specific Bmem promises to offer a window into an individual’s exposure history and immune status beyond that gleaned from traditional serum antibody meas-urements alone. ImmunoSpot® has emerged as a suitable technique for the de-tection of individual antigen-specific B cells through visualizing their anti-body-derived secretory footprints. Here, we report the theoretical and practical foundations for detecting rare antigen-specific Bmem in human peripheral blood mononuclear cells (PBMC). Leveraging the unique availability of verifiably naïve vs. antigen-experienced human samples, we used SARS-CoV-2 Spike (S-) and Nucleocapsid (NCAP) antigens to interrogate the presence of Bmem with these re-spective specificities. While 100% diagnostic accuracy was achieved for both an-tigens, detection of NCAP-specific Bmem required extension of the lower detection limit (LOD) of the standard assay. This could be achieved using 2 million PBMC in aggregate. The protocols described here should facilitate reliable detection of ASC present at varying precursor frequencies and serve as guidance for routine im-mune monitoring of rare Bmem with specificity for any antigen.