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Analyse IFNg and IL17A with ELISPOT vs FluroSpot

Guest Sara

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I am interested in transitioning from ELISPOT to FluoroSpot. I currently analyze T-cell activation in antigen-stimulated PBMCs with ELISPOT for IFNg and IL17A. It would be nice to change to FluoroSpot, and get the information on double secreting cells, but I am worried that there may be a difference in the result of cells producing single cytokines. Do you have any experience with this? I am also interested in the triple FluoroSpot for IFNg/IL17A/IL22, if it is compareable to my ELISPOT assays.  



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  • 2 weeks later...

Hello Sara,


In transitioning to FluoroSpot from the ELISpot assay there are two important factors one should be aware of when it comes to comparing absolute spot-numbers between the two assays.


1. Capture effects and the inclusion of anti-CD28 antibody.

2. Sensitivity due to camera settings.



What do I mean by the first point? Well, capture effects are an inherent part of the FluoroSpot assay since each well is coated with two or more anti-cytokine capture antibodies. For the majority of cytokine combinations that we have tried, this reduction in free cytokine does not present a problem, but in the case of IL-2 it will. Thus, capture of IL-2 will influence not only the IFN-g response but also that of some other cytokines (e.g. IL-4 and IL-5). In the FluoroSpot assay, we have solved this issue by including a costimulatory anti-CD28 antibody which effectively compensates for this capture of IL-2. This antibody is however also included in the IFN-gamma/IL-17A FluoroSpot kit, in this case not to compensate for capture but for a general strengthening of the cellular response. Thus, if you compare a single coated ELISpot well for IL-17A without anti-CD28 with that of dual coated IFN-g/IL-17A FluoroSpot well including anti-CD28 during stimulation, experimental conditions will be different, and spot-numbers could mismatch slightly. 


In general, capture effects have been carefully investigated for both IFN-g and IL-17A in a recent publication made by some very good people here at Mabtech R&D: Dillenbeck et al. Cells 2014, 3, 1116-1130.pdf 


Herein, comparisons have been made between single-coated and triple-coated FluoroSpot wells (IFN-g/IL-22/IL-17A). In addition, the sensitivity is compared to that of ELISpot. A good publication to read through!




In all of Mabtech's marketing material and in the publication attached above, we state that sensitivity of the FluoroSpot assay is similar to that of ELISpot. Although this statement is true it will ultimately depend on how you configure the camera settings of your FluoroSpot reader.


Thus, the reader controls how much of the fluorescent light is captured by the camera system during reading. In the world of photography this is referred to as "Exposure". For example, in the FluoroSpot reader manufactured by AID, different exposure settings can be manually put in by the user together with something called “Gain”. These two parameters (Exposure+ Gain) combined will result in the final digital image that is then sent to the software's counting algorithm where spot-numbers are finally determined. Due to this, the number of detectable spots in the AID reader is very much dependent on how you set these two values, Exposure and Gain.


A rather high setting here will result in the reader capturing a lot of light from the analyzed FluoroSpot wells and sensitivity is similar to that of ELISpot. By contrast, a low setting for Exposure/Gain, and the counting algorithm of the reader will simply not see all spots. For clarification on how this works I have attached a powerpoint file where the same well of an IL-6 FluoroSpot has been analyzed using three different camera settings for Exposure and Gain. Despite using exactly the same count settings in all three cases, spot-numbers will vary from 181 to 254 for the same well. 


Take a look in the powerpoint attachment below and it will make a lot more sense! 




Our triple FluoroSpot IFN-gamma/IL-22/IL-17A is a great new product in Mabtech's portfolio that we are really excited about! The analysis simply looks fantastic in a reader that can properly handle all three filters (FITC, Cy3 and Cy5). Sitting together in the reader room I often refer to the experience of analyzing a triple plate as the "Revenge of the Nerds". You can see examples of how this can look in the Cells publication attached above. These kits only come in a pre-coated format as we want to make sure that the customer gets the best possible result by taking away the "tricky" part of having to do the plate-coating process themselves. 




Finally, you or anyone else for that matter, should contact us directly to try out this FluoroSpot kit in your own laboratory. Mabtech can help with FluoroSpot reading by having the plates sent to us after the assay has been run! They are actually quite stable and can easily be transported for analysis.






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