Four-Color ImmunoSpot Assays Requiring Only 1-3 mL of Blood Permit Precise Frequency Measurements of Antigen-Specific B Cells-Secreting Immunoglobulins of All Four Classes and Subclasses.

The B lymphocyte response can encompass four immunoglobulin (Ig) classes and four IgG subclasses, each contributing fundamentally different effector functions. Production of the appropriate Ig class/subclass is critical for both successful host defense and avoidance of immunopathology. The assessment of an antigen-specific B cell response, including its magnitude and Ig class/subclass composition, is most often confined to the antibodies present in serum and other biological fluids and neglects monitoring of the memory B cell (B) compartment capable of mounting a faster and more efficient antibody response following antigen reencounter.

Assessing the Affinity Spectrum of the Antigen-Specific B Cell Repertoire via ImmunoSpot.

The affinity distribution of the antigen-specific memory B cell (B) repertoire in the body is a critical variable that defines an individual's ability to rapidly generate high-affinity protective antibody specificities. Detailed measurement of antibody affinity so far has largely been confined to studies of monoclonal antibodies (mAbs) and are laborious since each individual mAb needs to be evaluated in isolation. Here, we introduce two variants of the B cell ImmunoSpot assay that are suitable for simultaneously assessing the affinity distribution of hundreds of individual B cells within a test sample at single-cell resolution using relatively little labor and with high-throughput capacity.

Monitoring Memory B Cells by Next-Generation ImmunoSpot Provides Insights into Humoral Immunity that Measurements of Circulating Antibodies Do Not Reveal.

Memory B cells (B) provide the second wall of adaptive humoral host defense upon specific antigen rechallenge when the first wall, consisting of preformed antibodies originating from a preceding antibody response, fails. This is the case, as recently experienced with SARS-CoV-2 infections and previously with seasonal influenza, when levels of neutralizing antibodies decline or when variant viruses arise that evade such. While in these instances, reinfection can occur, in both scenarios, the rapid engagement of preexisting B into the recall response can still confer immune protection.

Antibody Levels Poorly Reflect on the Frequency of Memory B Cells Generated following SARS-CoV-2, Seasonal Influenza, or EBV Infection.

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.

Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays.

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.