Four color B cell immunospot assays for simultaneous detection of all four immunoglobulin classes and subclasses

Introduction ELISPOT is the only technique currently available that permits to enumerate antigen-specific B cells providing insights into humoral immune defense. Resting memory B cells can be detected in blood samples after polyclonal stimulation. In contrast, the spontaneous production of immunoglobulins (Ig) by B cells in freshly-isolated peripheral blood mononuclear cells (PBMC) signifies recent and ongoing antigen stimulation in vivo. ELISPOT has thus far been restricted to single color analysis of Ig classes or subclass for detection of Antibody-Secreting Cells. Consequently, a comprehensive study of all four Ig classes or IgG subclasses required fourfold amounts of PBMC, materials and labor. We have developed a 4-color B cell ImmunoSpot assay where all four Ig classes or IgG subclasses are detected simultaneously. This novel technique was applied to the characterization of Human Cytomegalus Virus (HCMV)-specific Ig responses in human subjects with previous exposure to the virus.