Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay

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.

Figure 1

Correlation between antibody-secreting cell counts obtained with the traditional ELISpot and the novel B cell fluorospot assay. IgG ASCs (on the left) and IgM ASCs (on the right) counted with the ELISpot assay are plotted on the x-axis and those counted with the fluorospot assay in side-by-side experiments are plotted on the y-axis.

Figure 2

Simultaneous detection of IgG and IgM antibody-secreting cells with a multiplex B cell fluorospot. IgG-7B2 and IgM-2B9 cells were mixed at various ratios (1:0, 1:0.5 and 1:1, as described in the text) and incubated in anti-IgG+M-coated wells. The plot shows the IgG (in red) and IgM (in blue) ASC frequency in each of duplicated wells of two experiments (▼ and ▲) relative to the number of spots counted in wells in which only one cell line was cultured. Dotted lines show the expected values at different cell ratios. Representative wells are shown.

Figure 3

Muliplex detection of total or HIV-1 gp41 Env-specific IgG and IgM antibody-secreting cells. IgG-7B2 and IgM-2B9 cells were co-cultured using either HIV-1 MN gp41 Env (top picture) or a mixture of anti-human IgG and IgM mAbs (bottom picture) as capture reagents to detect IgG-secreting (in red) and IgM-secreting (in blue) ASCs. The graph on the right shows no statistically significant difference in the numbers of ASC/well detected using the two B cell fluorospot assay formats (n = 2).