Abstract
The traditional enzyme-linked immunospot (ELISpot) assay is the gold standard for the enumeration of antigen-specific B cells. Since B cell availability from biological samples is often limited, either because of sample size/volume or the need of performing multiple analyses on the same sample, the implementation of ELISpot assay formats that allow the simultaneous detection of multiple antibody types is desirable. While dual-color ELISpot assays have been described, technical complexities have so far prevented their wide utilization as well as further expansion of their multicolor capability. An attractive solution is to replace the chromogenic reaction of the traditional ELISpot assay with a fluorescent detection system (fluorospot assay). Fluorospot assays using fluorophore-conjugated secondary antibodies in conjunction with fluorescence enhancers, FITC/anti-FITC and biotin/avidin amplification systems and dedicated equipment for spot detection have been developed to enumerate T-cells secreting two or three specific cytokines and, more recently, IgG and IgA antibody-secreting cells (ASCs). We hereby report a method for a multiplex B cell fluorospot assay that utilizes quantum-dot nanocrystals as reporters without further amplification systems or need of dedicated equipment. With this method we simultaneously enumerated HIV-1 gp41 envelope glycoprotein-specific IgG and IgM antibody-secreting cells with sensitivity comparable to that of the traditional ELISpot assay.
Highlights
The gold standard for the enumeration of immune cells secreting a specific protein, either antibodies or cytokines, is the enzymed-linked immunospot (ELISpot) assay
We first evaluated the sensitivity of the B cell fluorospot assay in a single-color format using the traditional B cell ELISpot assay as control
IgG-7B2 and IgM-2B9 cells—two immortalized B cell hybridoma cell lines secreting anti-HIV-1 gp41 envelope glycoprotein (Env) IgG and IgM mAbs, respectively—were plated at cell densities ranging from 100 cells/well to 5000 cells/well and antibody-secreting cells (ASCs) were detected by using anti-IgG
Summary
The gold standard for the enumeration of immune cells secreting a specific protein, either antibodies or cytokines, is the enzymed-linked immunospot (ELISpot) assay. The ELISpot assay is widely used to enumerate antibodies secreted by B cells [3,4,5] and cytokines secreted by. The traditional ELISpot assay has been further modified by replacing the chromogenic reaction with fluorophore-conjugated antibodies (fluorospot assay) for the simultaneous detection of two [9,10,23] and three [12] proteins secreted by T cells. As novel uses of the flurospot assay and exciting implementations of detection systems and data analysis are being developed, we hereby describe a modified method for the B cell fluorospot assay in which fluorophores are replaced with quantum-dot nanocrystals to simultaneously detect antigen-specific
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