many spots in negative control

[Guest Janou]

We have use your pre-coated IFN-g Elispot plates (3420-4AST plates and strips) and have noticed an unusual phenomena that I'm hoping you can help me out with.

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We are running human PBMCs in duplicate of sarcoïdosis patients. Rows 1 and 2 contain negative control, rows 3-10 contain our test antigens and rows 11 and 12 contain the positive control. We have noticed that for some patients the amount of spots in the negative control is very high (sometimes even higher than the wells containing a tested antigen), making it impossible to define whether or not that patient has a positive reaction to one of the tested antigens. Every row contains 2.0 *10^5 cells, so that should not be the cause of this phenomenon. We have now tested 150 patients and we see that the amount of spots in the negative control seems to increase even more as we continue our test. We do not know what causes this problem and how we can repair it. 

hopefully you can help us

[Guest Janou]

perhaps it is practical to add that the negative control consists of AIM-V medium and cells of a sarcoïdosis patients.

[Christian@mabtech.com]

Dear Janou,

Welcome to the forum. I have a some feedback:

IFNg elispot does not always result in a very low background (0-5 spots/well). If you run 50 healthy donors you will probably have 90% very low back background, 5% moderate background and 5% high background. With 250,000 cells/well, I would say moderate background is 10-15 spots/well and high background is 25-35 spots/well. Factors such as isolation technique, cell culture medium, serum used and the sensitivity of your ELISpot assay will all affect these numbers. Furthermore, the count settings of you ELISpot readers is greatly, greatly influential. 

Why do some totally healthy donors have a lot of background spots? I do not know for sure, but my speculation is that the "immune status" naturally plays a role in these background numbers. Some might be fighting off a "spur" of CMV infection somewhere in the body. This happens all the time (and without any symptoms) and could in theory generate a higher background of IFNg in unstimulated PBMC. But there could also be a factor of outside stimulation like isolation technique and bad serum.   

Patients will naturally have a much more complicated "immune status" compared to healthy donors, so it is not surprising that background levels tend to go up. Furthermore, one should not forget that NK-cells also have the capacity to secrete IFNg. Maybe these cells are more activated in the blood stream of sarcoïdosis patients, which could be an alternative explanation to the background observed. 

Looking at you wells it does look like well F6 in the second plate has a lower number of spots compared to the unstimulated F2. I would here say two things:

- In F6 you have incubated PBMC with antigen. Although no antigen specific T-cells have been activated, the T-cells could still be affected differently by the antigen added. Monocytes could take up the antigen, process it and release cytokines which fundamentally changes the cytokine environment of the well. We know from experience that many antigens are contaminatied with TLR-ligands. These monocyte derived cytokines could in turn downregulate the T-cells/NK-cells secreting IFNg directly exvivo. This could be one explanation for the phenomenon observered.  

- You reader says there are 46 spots in well F6 and 90 spots in the unstimulated control F2. These numbers will naturally be very dependent on the settings of your AID reader. Maybe you have the count settings rather high, meaning you only count rather dark and bigger spots? In well F6 you might in reality have 80 spots, but only 46 are counted by your settings due the high threshold you have applied. Something to look into. Take the F row as an example, here I would say that only well F6 falls below what is considered natural variability for an ELISpot assay. With a different count setting the difference could even be smaller?

We have made a tutorial on the AID count settings on youtube. Can be found here:

 

Or even better, consider switching to our next generation IRIS reader. With this reader I really recommend switching over to FluoroSpot, even for single color IFNg analysis. The RAW technology makes life so much easier and the RSV values are increadibly fun to analyze: