Technote: Measuring amount of secreted analyte (RSV)

Published: November 23, 2022

Updated: November 23, 2023

The spot counting algorithm of Mabtech IRIS extracts a 3D-volume for each spot in a FluoroSpot assay, corresponding to the relative amount of secreted analyte. This previously unattainable dimension of spot data is called relative spot volume (RSV).


Our aim of the present study was to investigate whether RSV analysis can be used as a tool for a more detailed characterization of T cell responses, as a complement to spot number analysis.



To address the question at hand, we compared spot frequencies and RSV values of CD4+ T cell responses induced by the mitogen ConA or by immunization with a peptide containing an MHC class II I-Ad restricted epitope. T cell responses were analysed using an IFN-γ/IL-2 FluoroSpot (Fig. 1).

Despite showing similar double-positive IFN-γ/IL-2 spot numbers (pink bars in Fig. 1), the RSV values of IFN-γ as well as IL-2 in these double-positive spots were higher after peptide stimulation compared to ConA stimulation (Fig. 2).

The higher RSV value of a spot corresponds to the higher intensity and larger size of the spot (Fig. 3). In addition, the RAWspot algorithm takes into account diffusion patterns to simulate the point of origin and the full relative volume of the spot.



BALB/c mice were immunized three times subcutaneously with 100 μg peptide together with an ISCOM-based adjuvant followed by an intraperitoneal boost with 100 μg peptide in PBS. Spleens were harvested eight days after the final boost. Splenocytes were prepared and stimulated with 2 μg/ ml peptide or 5 μg/ml ConA and analysed using IFN-γ/IL-2 FluoroSpot.



  • Immunization with peptides induced T cells that secreted more cytokine per cell than mitogen-stimulated cells.
  • Combining spot numbers with RSV values enables more precise characterization of antigen-induced T cell responses.
RSV graph

Fig. 1 Splenocytes stimulated with peptide or ConA and analyzed using IFN-γ/IL-2 FluoroSpot. Bar-graph shows spots from single- and double secreting cells.

RSV treegraph

Fig. 2 RSV-data from the double-positive IFN-γ/IL-2 spots. Dot-plot depicts relative amount of secreted IFN-γ or IL-2 in two representative wells.

RSV close-up

Fig. 3 White arrows indicate double-positive IFN-γ/IL-2 spots produced by splenocytes.

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