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.
Highlights
There are many assays designed to measure antibody reactivity and specificity (e.g., ELISA, immunoblot, etc.) but only a few focuses directly on the antibody-secreting cells (ASC)
If B cells have been potently activated in vivo and have developed into ASC, they can be added to an ELISpot assay plate and incubated in cell culture medium without any additional stimulation
We found that the FluoroSpot technique was as sensitive as the B-cell ELISpot run in parallel and offered the additional possibility to define and enumerate, in a single well, B cells secreting antibodies of both IgG and IgA isotype
Summary
There are many assays designed to measure antibody reactivity and specificity (e.g., ELISA, immunoblot, etc.) but only a few focuses directly on the antibody-secreting cells (ASC). We have in this study evaluated a newly developed B-cell FluoroSpot assay for the analysis of human IgG- and IgA-secreting B cells activated in response to Influenza A (H1N1) vaccine (Pandemrix®). We found that the FluoroSpot technique was as sensitive as the B-cell ELISpot run in parallel and offered the additional possibility to define and enumerate, in a single well, B cells secreting antibodies of both IgG and IgA isotype. This makes the technique suitable in situations where the source of cells or the amount of Ag is limited. Relevant application areas for the methods include detection of B-cell responses in various diseases and those elicited by vaccination
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