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