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A Comparison of Flow Cytometry-based versus ImmunoSpot- or Supernatant-based Detection of SARS-CoV-2 Spike-specific Memory B Cells in Peripheral Blood

Lookup NU author(s): Georgia Stylianou, Dr Sharon CooksonORCiD, Dr Stephen Todryk

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).

Abstract

© 2025 by the authors.Background: 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. Methods: 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 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+ Bmem 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 Bmem in antigen-primed individuals when such Bmem occur in the mid- to high-frequency range, and that they broadly concur. Strengths and weaknesses of the three test systems are discussed.

Publication metadata

Author(s): Stylianou G, Cookson S, Nassif JT, Kirchenbaum GA, Lehmann PV, Todryk SM

Publication type: Article

Publication status: Published

Journal: Vaccines

Year: 2026

Volume: 14

Issue: 1

Online publication date: 24/12/2025

Acceptance date: 21/12/2025

Date deposited: 16/02/2026

ISSN (electronic): 2076-393X

Publisher: Multidisciplinary Digital Publishing Institute (MDPI)

URL: https://doi.org/10.3390/vaccines14010020

DOI: 10.3390/vaccines14010020

Data Access Statement: The data generated in this study will be made available by the authors, without undue reservation, to any qualified researcher

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