The traditional enzyme-linked immunospot (ELISpot) assay is the gold standard\r\nfor the enumeration of antigen-specific B cells. Since B cell availability from biological\r\nsamples is often limited, either because of sample size/volume or the need of performing\r\nmultiple analyses on the same sample, the implementation of ELISpot assay formats that\r\nallow the simultaneous detection of multiple antibody types is desirable. While dual-color\r\nELISpot assays have been described, technical complexities have so far prevented their wide\r\nutilization as well as further expansion of their multicolor capability. An attractive solution\r\nis to replace the chromogenic reaction of the traditional ELISpot assay with a fluorescent\r\ndetection system (fluorospot assay). Fluorospot assays using fluorophore-conjugated\r\nsecondary antibodies in conjunction with fluorescence enhancers, FITC/anti-FITC and\r\nbiotin/avidin amplification systems and dedicated equipment for spot detection have been\r\ndeveloped to enumerate T-cells secreting two or three specific cytokines and, more recently,\r\nIgG and IgA antibody-secreting cells (ASCs). We hereby report a method for a multiplex\r\nB cell fluorospot assay that utilizes quantum-dot nanocrystals as reporters without further\r\namplification systems or need of dedicated equipment. With this method we simultaneously\r\nenumerated HIV-1 gp41 envelope glycoprotein-specific IgG and IgM antibody-secreting\r\ncells with sensitivity comparable to that of the traditional ELISpot assay.
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