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

Cells. 2021 Jul 21;10(8):1843. doi: 10.3390/cells10081843.


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.

Keywords: EBV; HCMV; SARS-CoV-2; antibody-secreting cell; antigen coating; fluorospot; immune monitoring; influenza; memory B cell; plasmablast.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Viral / analysis*
  • B-Lymphocytes / immunology*
  • COVID-19
  • Enzyme-Linked Immunospot Assay / methods*
  • Histidine
  • Humans
  • Immunologic Memory*
  • Mice
  • Oligopeptides
  • SARS-CoV-2 / immunology*
  • SARS-CoV-2 / metabolism
  • Viral Proteins / immunology*


  • Antigens, Viral
  • His-His-His-His-His-His
  • Oligopeptides
  • Viral Proteins
  • Histidine