Comparing the diagnostic specificity of flow cytometry-based probe staining and ELISPOT for detection of SARS-CoV-2 Spike-reactive memory B cells using defined cohorts of cryopreserved human PBMC 9313

Abstract Description Determining if an individual has acquired immunity, whether it be against an infectious agent or an innocuous environmental or food allergen, in the clinical diagnostic realm relies nearly exclusively on the detection of antibody reactivity. However, there is accumulating evidence indicating that assessment of memory B cell (Bmem) reactivity provides more reliable information on the current state of humoral immunity than measurements of serum antibody reactivity alone. There are two prevalent techniques that are suitable for detecting rare antigen Bmem at single-cell resolution: flow cytometry-based antigen probe staining and ELISPOT. Antigen probe staining involves surface labeling of single cell suspensions with fluorescently-tagged antigen probe(s) and Bmem with sufficient affinity for the antigen probe(s) acquire fluorescence and can be further characterized based on their surface phenotype. Alternatively, ELISPOT enables identification of antigen-specific Bmem through detection of their antibody-derived secretory footprints. Utilizing cryopreserved human PBMC samples originating from defined cohorts, we systemically compared both techniques for their a) diagnostic specificity, b) cell material and labor requirements and c) reproducibility when samples were tested twice in a blinded fashion for (Bmem) reactivity against the SARS-CoV-2 Spike protein. The suitability of these techniques for immune monitoring of Bmem reactivity will be discussed and results presented. Funding Sources R&D Budget of CTL Topic Categories Translational and Interventional Immunology (TI)