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Extending ImmunoSpot® Assays' Sensitivity for Detecting Rare Antigen-Specific B Cells to One in a Million-And
Greg A Kirchenbaum1, Noémi Becza1, Lingling Yao1
1Research and Development, Cellular Technology Ltd. (CTL), Shaker Heights, OH 44122, USA.
Background/Objectives: Despite clonal expansion during a primary immune response, or after subsequent antigen encounters, the frequency of memory B cells (Bmem) specific for an antigen remains low, making their detection difficult. However, unlike serum antibodies, which have a short half-life in vivo and thus require continuous replenishment to maintain stable titers, circulating Bmem are long-lived; they preserve immunological preparedness through their ability to rapidly engage in recall responses and differentiate into antibody-secreting cells (ASCs) upon antigen encounter. To this end, development of assays suited for the reliable detection of rare antigen-specific Bmem is critical and can provide insights into an individual's antigen exposure history and immune status beyond that offered by traditional serum antibody measurements alone. Methods: ImmunoSpot® has emerged as a suitable technique for the detection of individual antigen-specific B cells through visualizing their antibody-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 respective specificities. Results: While 100% diagnostic accuracy was achieved for both antigens, detection of NCAP-specific Bmem required reducing the lower detection limit of the standard assay. Specifically, this was achieved by testing a total of 2 million PBMC across multiple replicate assay wells and assessing the cumulative number of secretory footprints detected. Conclusion: The protocols described here should facilitate the reliable detection of ASCs present at varying precursor frequencies and serve as guidance for routine immune monitoring of rare Bmem with specificity for any antigen.
Background/Objectives: Despite clonal expansion during a primary immune response, or after subsequent antigen encounters, the frequency of memory B cells (Bmem) specific for an antigen remains low, making their detection difficult. However, unlike serum antibodies, which have a short half-life in vivo and thus require continuous replenishment to maintain stable titers, circulating Bmem are long-lived; they preserve immunological preparedness through their ability to rapidly engage in recall responses and differentiate into antibody-secreting cells (ASCs) upon antigen encounter. To this end, development of assays suited for the reliable detection of rare antigen-specific Bmem is critical and can provide insights into an individual's antigen exposure history and immune status beyond that offered by traditional serum antibody measurements alone. Methods: ImmunoSpot® has emerged as a suitable technique for the detection of individual antigen-specific B cells through visualizing their antibody-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 respective specificities. Results: While 100% diagnostic accuracy was achieved for both antigens, detection of NCAP-specific Bmem required reducing the lower detection limit of the standard assay. Specifically, this was achieved by testing a total of 2 million PBMC across multiple replicate assay wells and assessing the cumulative number of secretory footprints detected. Conclusion: The protocols described here should facilitate the reliable detection of ASCs present at varying precursor frequencies and serve as guidance for routine immune monitoring of rare Bmem with specificity for any antigen.
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