B cell ELISpot enables a sensitive analysis of antigen-specific B cells at the single cell level but is limited to the analysis of reactivity with a single antigen. By reversing the B cell ELISpot and using anti-IgG capture antibodies instead of coated antigen, the specificity of antibodies secreted by B cells can be defined using soluble tagged antigen for detection. When combining this approach with fluorescent detection of the antigen in a B cell FluoroSpot assay, reactivity with multiple antigens can be defined. In the protocol described herein, splenocytes from a mouse immunized with an antigen were analyzed for their reactivity with the antigen used for immunization and for cross-reactivity with a different but structurally related antigen. Using this assay, we found that at least 15% of the B cells displayed detectable cross-reactivity. B cell FluoroSpot utilizing multiple antigens provides a tool for a single-cell analysis of B cell cross-reactivity, for example, with variable and polymorphic antigens found in various pathogens; or analysis of other types of immune responses where analysis of cross-reactivity is of interest. It is also possible to simultaneously analyze B cell reactivity to completely different antigens.
Keywords: Antibody; B cell; Cross-reactivity; ELISpot; FluoroSpot; Peptide tag.