Before FluoroSpot, there was a suboptimal method for multiplexing called dual color ELISpot. In this setup, two different substrates were cleaved with two different enzymes, giving rise to two colors.
We consider the method suboptimal, because the two enzymatically developed colors can mask each other, leading to unreliable results as found by Janetzki et al. 2014. If, for example the “red analyte” is secreted in low amounts, whereas the blue spot is massive, the cell that secreted both may incorrectly be counted as a single-analyte-secreting cell (see image). In addition, the two substrate reactions need to be performed sequentially and may interfere with one another, also leading to erratic results.
Needless to say, we didn’t find this method to be effective or accurate, and strongly refrain from using it. Instead, we recommend using FluoroSpot – a reliable assay where each fluorophore is detected using separate filters. This allows for an accurate way of multiplexing, leading to better results.
Dual ELISpot is biased towards one color.