Comparison of ELISpot and FluoroSpot in the Analysis of Swine Flu-Specific IgG and IgA Secretion by in Vivo Activated Human B Cells

Abstract

We have evaluated a novel B-cell FluoroSpot assay for the analysis of antibody responses in healthy individuals vaccinated intramuscularly with Influenza A (H1N1) antigen (Pandemrix®, GlaxoSmithKline). Using the FluoroSpot assay and an ELISpot assay run in parallel for comparison, we measured the frequency of cells secreting antigen-specific as well as total IgG or IgA antibodies seven days post vaccination. The assays were based on high affinity monoclonal antibodies for capture and detection of human IgG and IgA. Whereas conventional ELISpot analyzes IgG- and IgA-secreting B cells separately, fluorescent detection enabled simultaneous enumeration of B cells secreting IgG or IgA in the same well. The FluoroSpot protocol was also simpler as the assay could be performed without the need for an amplifying detection step. While having all the advantages of a conventional ELISpot assay, including high sensitivity, robustness and ease of performance, the FluoroSpot assay adds further value in reducing costs, time and material.

Figure 1

(a) Analysis of Influenza A (H1N1)-specific IgG- and IgA-secreting cells in ELISpot and in FluoroSpot. PBMC were collected before and seven days after vaccination with the influenza vaccine Pandemrix^®. Ag-specific IgG- and IgA-ASC were detected using 0.75 μg/well of coated Ag. After overnight culturing of PBMC (250,000 cells/well), detection mAbs specific for IgG and IgA, and labeled with different fluorophores, were mixed together and added to the wells (FluoroSpot). In ELISpot, ASC were detected by adding biotin-labeled anti-IgG or anti-IgA detection mAbs followed by SA-ALP and a precipitating substrate. The figure shows representative examples of the vaccine-induced responses in donor 1; (b) Analysis of Influenza A (H1N1)-specific IgG- and IgA-secreting B cells in ELISpot and in FluoroSpot in six donors (ASC/ 250,000 PBMC). No Ag-specific ASC were seen before vaccination. Grey bars represent ELISpot whereas colored bars represent FluoroSpot. The diagram shows mean values with SD (n = 3). Each set of bars corresponds to data from donor 1–6 in numerical order from left to right.

Figure 2

(a) Analysis of the total number of B cells secreting IgG and IgA after vaccination with Pandemrix^®. The wells were coated with mAbs specific for IgG and/or IgA. After an overnight culture of PBMC (100,000 cells/well) the detection procedure was identical to that used for detection of Ag-specific ASC in Figure 1a. The images are representative examples from donor 1. (b) ELISpot and FluoroSpot analysis of the frequency of B cells secreting IgG and IgA before and after vaccination with Pandemrix^® (ASC/100,000 PBMC). The diagram shows mean values with SD (n = 3). Each set of bars corresponds to data from donor 1–6 in numerical order from left to right.