A comparison of Flow Cytometry- versus ImmunoSpot- or Supernatant-Based Detection of SARS-CoV-2 Spike-Specific Memory B Cells in Peripheral Blood

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.