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Guest Flavia

False positve in IFN gamma ELISpot

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Guest Flavia

Hello this is Flavia,

I am performing an IFN gamma ELIspot (pulse incubation with peptide) using cryopreserved splenocytes obtained from mice that were primed-boosted (exp.1) or only primed (exp.2) in vivo with intravenous injection of Adenovirus.  The splenocytes were cryopreserved in Fetalclone III serum and DMSO.

Before running all my experiential points, I did a pilot run to optimize peptide concentration and incubation time (24h and 48h). I used 1million splenocytes/well as reported in the two article I used as reference for synthetizing the peptide. For the assay I used RPMI1640, 10%HI-FBS, L-Glu, Pen/Strep, 50uM BME, IL-2 10U/ml, and I run 4 samples, control saline and Adenovirus, for both exp.1 and 2.

A strange thing happened. For the exp.2 samples I obtained exactly the results I was expecting (ctrl saline was negative and Adenovirus was nicely positive). For exp.1 instead I obtained positive spots for both ctrl saline and Adenovirus, with the Adenovirus sample presenting an higher number of spots.

To try to solve this false positive issue I am planning to: using AIM V  medium, not adding IL-2, seeding less cells (250.000 and 500.000 cells/well) incubate 16-18h, and run the same samples stimulated and not-stimulated.

Do you think I should add some more conditions?

Thank you in advance. Regards,

Flavia

P.S. I order the item 3321-4HST-2 that was supposed to have transparent PVDF membrane, but the membrane is instead white.

 

 

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Hi Flavia and welcome to the Mabtech Forum!

I think you are addressing the issue you're having in the best possible way. 

My guess is that it is the added IL-2 that is causing you false-positive background, so in my view it's good that you are removing it. You shouldn't need extra IL-2 for a short incubation like this anyway. Did you include it in your original protocol for a particular reason?

The 1 million splenocytes/well is on the brink of being too many, so you're doing the right thing of decreasing that number to 250k and 500k cells/well.

If you didn't have the no-stimulation-condition before, it's good that you have chosen to include that negative control now. 

Incubating for a shorter time, e.g. your suggested 16h-18h is also wise. The kinetics of IFN-gamma secretion is quite fast, and although you might catch a few extra responding cells at 24h, you also increase the risk of background. 

Whether to use serum-free AIM-V or RPMI1640 supplemented with FBS I don't think it will make much of a difference. We always use RPMI+FBS in-house, but then again we have tight control of the FBS we are using. If you are unsure of the quality of your FBS, please go ahead and use AIM-V instead. In our hands, that is the serum-free medium that works the best.

Finally, the words "transparent" (or "clear) and "white" in the plate names refer to the plastic of the plate, not the PVDF membrane. The membrane is always white. You can see the difference in plastic of two different MSIP-plates, transparent and white (your strip-plate is similar to the transparent one), in this video at approx. 50 seconds:

 

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Guest Flavia

Dear Jens,

thank you very much for your answer! As soon as I received the AIM V media I will let you know the outcome of my second pilot run.

I am new to immunological assays and I thought that IL-2 was always required in the restimulation of T cells ex vivo so I included it.

Regards,

Flavia

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Guest Flavia

Hello, I am back with updates.

I have just finished the second pilot using AIM V media and omitting IL-2. I tested 3 different cell dilutions (100-250-500k) and I incubated with OR without the peptide for 24 hours.

As far as the "false positive" issue I think I solved the problem.

Unfortunately I have a new one. I have some unspecific INFg secretion in the samples from the treated groups (Adenovius injected) that I incubated without the peptide. Do you think it depends on the number of cells? 

Thank you in advance.

FLAVIA

Picture1.tif

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Hi Flavia,

Sorry for the delay in response, you caught us in the middle of our midsummer's holiday. 

So, before in the experiment with primed+boosted mice you saw false positive spots in the "Ctrl vh +pept" wells, which are now gone. But instead now you have spots in the "Ad-GFP -pept" wells, a type of control you didn't include in your first run, correct?

In essence, it looks like the T cells are secreting IFNg without the need for peptide re-stimulation. Could that be a true finding? Yes, it is not surprising. With the prime and the boost you are stimulating the T cells in vivo, and many of the cells are probably still activated and secreting IFNg even after splenectomy, cryopreservation and thawing. I don't know how long after prime or boost you are taking the spleens, but if we are talking days after immunization then what you see is most probably correct. We see this from time to time, especially in the context of Adenovirus which usually gives the T cells a real jolt in terms of IFNg-production. As you can see in your picture, the peptide re-stimulation thus however increase the number of spots, at least in exp 1. It's great to see also that you have solved the issue with false positive spots in the "Ctrl vh + pept" wells. 

//Jens

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