The scope of immune monitoring is to define the existence, magnitude, and quality of immune mechanisms operational in a host. In clinical trials and praxis, the assessment of humoral immunity is commonly confined to measurements of serum antibody reactivity without accounting for the memory B cell potential. Relying on fundamentally different mechanisms, however, passive immunity conveyed by pre-existing antibodies needs to be distinguished from active B cell memory. Here, we tested whether, in healthy human individuals, the antibody titers to SARS-CoV-2, seasonal influenza, or Epstein–Barr virus antigens correlated with the frequency of recirculating memory B cells reactive with the respective antigens. Weak correlations were found. The data suggest that the assessment of humoral immunity by measurement of antibody levels does not reflect on memory B cell frequencies and thus an individual’s potential to engage in an anamnestic antibody response against the same or an antigenically related virus. Direct monitoring of the antigen-reactive memory B cell compartment is both required and feasible towards that goal.

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general.