Multiplex ImmunoSpot ® Assays for the Study of Functional B Cell Subpopulations.

B cells mediate humoral immunity by producing antibody molecules, but they also participate in innate and acquired immune functions via the secretion of effector molecules such as cytokines, chemokines, and granzyme. B cell subpopulations releasing such effector molecules have been implicated in immunobiology and a number of diseases.Unlike antigen-specific T cells that can be identified by multimer staining, and then counter-stained to define T cell subpopulations, antigen-specific B cells cannot be detected by flow cytometry. Staining antigen-specific B cells with labeled antigen, in large, has been unsuccessful. Instead, antigen-specific B cells can be and are commonly studied by ELISPOT. In the ELISPOT approach, the B cell is identified via the antibody that it secretes being captured on a membrane by the antigen itself. Should it be feasible to measure simultaneously antibody production and the secretion of other secretory B cell products, it would then be possible to identify B cell subpopulations that co-express effector molecules. Here we introduce multiplex ELISPOT assays in which measurements of antibody secretion are combined with the detection of Granzyme B, IL-6, IL-10, IFN-γ, and TNF-α. Such multiplex assays will help define effector B cell subpopulations, as well as the understanding of their role in health and disease.