Memory B cells (Bmem) facilitate the generation of renewed and rapid antigen-specific antibody responses long after the initial antigen exposure, at a time when circulating serum antibodies may have declined. As the generation and/or recruitment of Bmem is at the core of most vaccination strategies, the assessment of antigen-specific Bmem is highly informative for forecasting and profiling the elicited B cell immune response. The two prevalent techniques used to detect antigen-specific Bmem cells at single cell resolution are probe-based flow cytometry and B cell ImmunoSpot, while the measurement of B cell-derived antibodies in culture supernatants of stimulated B cells offers a semi-quantitative alternative. To the best of our knowledge, a direct side-by-side comparison of these test systems has not yet been reported. Hence, in a blinded fashion, we compared these three test systems in parallel, aiming to detect SARS-CoV-2 Spike protein- (S-antigen) specific IgG+ Bmem in peripheral blood mononuclear cells (PBMC) samples obtained from well defined cohorts comprising: pre-COVID era “naïve” individuals (negative controls), individuals shortly after recovery from a PCR-verified SARS-CoV-2 infection (positive controls), and a cohort of donor PBMC isolated in 2024 (the experimental group). Our results demonstrate that all three assays are suited for the detection of specific Bmem in antigen-primed individuals when such Bmem occur in the mid- to high frequency range; strengths and weaknesses of the three test systems are discussed.

Background: Memory B cells (B_mem) facilitate the generation of renewed and rapid antigen-specific antibody responses long after the initial antigen exposure, at a time when circulating serum antibodies may have declined. As the generation and/or recruitment of B_mem is at the core of most vaccination strategies, the assessment of antigen-specific B_mem is highly informative for forecasting and profiling the elicited B cell immune response. Methods: The two prevalent techniques used to detect antigen-specific B_mem cells at single-cell resolution are probe-based flow cytometry and B cell ImmunoSpot, while the measurement of B cell-derived antibodies in culture supernatants of stimulated B cells offers a semi-quantitative alternative. To the best of our knowledge, a direct side-by-side comparison of these assay systems has not yet been reported using the same starting PBMC material in a blinded fashion to test all three assays simultaneously. Results: These three assay systems were run in parallel to detect SARS-CoV-2 Wuhan-1 strain Spike-specific IgG⁺ B_mem in peripheral blood mononuclear cell (PBMC) samples obtained from well-defined cohorts comprising pre-COVID-19 era “naïve” individuals (negative controls), individuals shortly after recovery from a PCR-verified SARS-CoV-2 infection (positive controls), and a cohort of donor PBMCs isolated in 2024 (the experimental group). Each assay was able to discern Spike-exposed individuals from naïve , with ImmunoSpot suggesting superior sensitivity and specificity. ImmunoSpot and flow cytometry results were closely correlated. Conclusions: The study demonstrates that all three assays are suited for the detection of specific B_mem in antigen-primed individuals when such B_mem occur in the mid- to high-frequency range, and that they broadly concur. Strengths and weaknesses of the three test systems are discussed.