Detecting all Immunoglobulin Classes and Subclasses in a Multiplex 7 Color ImmunoSpot^® Assay

Abstract

Antibody molecules in peripheral blood have a relatively short half-life of roughly 20 days, and therefore their persistence in the serum depends on continuous replenishment by plasma cells. Serum antibody titers are thus indirect and unreliable indicators of immunological memory. In contrast, memory B cells persist in peripheral blood for decades, and enumerating these cells provides direct evidence of having developed an immune response to a given antigen. ELISPOT is an ideal research tool for enumerating antigen-specific memory B cells. Traditionally, B cell ELISPOT assays have been performed for detecting a single class of immunoglobulin (Ig), using a single colorimetric substrate. For comprehensive monitoring of B cell memory, however, all immunoglobulin classes and subclasses need to be assessed. Thus, seven single color assays would need to be performed to measure the numbers of antigen-specific B cells producing IgM, IgA, IgE, IgG₁, IgG₂, IgG₃, and IgG₄. We report here the development of a multiplex seven color B cell ImmunoSpot^® assay in which the number of antigen-specific B cells can be established simultaneously for all major antibody classes and subclasses, requiring the PBMC, antigen, and labor corresponding to a single color assay.