Positive control for IL-12, IL-5 and IL-17A

[Questions from customers]

Dear Mabtech,

 

We try to see whether drug-specific IL-12p70- (and also IL-17-) releasing cells could be detected after incubating the culprit drug with PBMCs in patients with a confirmed history of drug allergy. We have successfully demonstrated drug-specific IFN-gamma and granzyme-B releasing cells after incubating drug with PBMCs for 48 hours in ELISpot plate, and 28 hours in 96-well plate/20 hours in ELISpot plate, respectively.  

 

We are quite happy with IFN-gamma and granzyme-B results. However, we have problems setting up IL-12p70 and IL-17 experiments (and to lesser extent, IL-5) as we rarely any spots even by stimulation with positive control (PHA). We normally incubate 250,000 cells/well with drug in ELISpot plate for 48 hours. We realize that you recommend using LPS+R848, not anti-CD3 or PHA, as a positive control for IL-12p70. Do we need to use R848 as a positive control along with LPS? 

 

Do you have any specific recommendation for the development of IL-12p70, IL17, (and IL-5) ELISpots in terms of incubation time or other special technique? How many spots do we expect to see in positive control well? As for granzyme B, we incubate the cells for 28 hours in 96 well-plate before moving into ELISpot for another 20 hours, is it possible to do the whole thing in ELISpot plate? Why do we need to do 2 steps, may I ask?

 

Best Regards, 

J

 

 

[Jens@mabtech.com]

Hi J,

 

Thank you for your questions!

 

Here are our thoughts:

 

1. PHA stimulates mainly lymphocytes, and IL-12 is generally produced by antigen presenting cells. Although there are some reports that T cells can be activated to produce IL-12, PHA is probably not the best stimulant in your assay. Along the same lines, anti-CD3 shouldn’t be an optimal stimulant for IL-12 either. We normally use LPS and LPS+IFNg as positive controls for IL-12(p70). I’m attaching some data from experiments where we have tried out different stimuli for IL-12(p40) and IL-23 on dendritic cells. Although these are purified DCs and not studied for release of IL-12(p70), the results might be relevant for your setting of PBMC and IL-12(p70). As seen in the attachment, LPS gives a modest stimulation whereas you get good induction if you combine LPS with R848 or IFNg (hiltonol = poly-lcic), and even better induction if you combine all three. As said, I’m not totally sure about how straight a parallel can be drawn to your setting, but as you suggest yourself, please try R848 in combination with LPS.

 

2. The IL-17A and IL-5 assays should give spots with PHA, even if the number normally is much lower than for IFNg. It’s good that you incubate for 48 hours since these two cytokines tend to have slow kinetics. Perhaps you have a weak PHA that works sub-optimally, and that in the case of IFNg is acceptable because you generally have many IFNg-spots, but in the case of IL-17A and IL-5 just isn’t strong enough? You could try to stimulate with anti-CD3 or even a combination of anti-CD3 and anti-CD28 instead, for all three analytes. 

 

3. I don’t recognise the recommendation to incubate in two steps, 28h+20h. In our data sheet, it only says that you should incubate for 48h in one single step in the ELISpot-plate. 

 

Looking forward to hearing from you again.

 

Kind regards,

Jens

 

IL12-23.pptx

[Questions from customers]

Hi Jens,

 

Thanks a lot for your reply. It definitely helps us guide the next experiments.

 

1. You bring up a very good point that IL-12 is mainly produced by antigen presenting cells. I assume that you refer to monocytes in PBMCs, or including B cells as well? Most of our ELISpot assays are performed with frozen PBMCs, which probably lead to B cell loss and alteration of dendritic cell subsets (Clin Exp Immunol. 2011 Jan;163(1):33-49). We can compare the results of IL12p70 stimulation in freshly isolated PBMCs vs. frozen PMBCs and let you know.

 

 2. We definitely can use anti-CD3 as a positive control for IL17 and IL5. It is good to know that IL17A needs long incubation time since we initially thought that it would show up fast. For any cytokines that happen to express much earlier, it is still OK if we leave cells on the plate longer than it should be during set up period, right? (may be too many spots I suppose) I remember that I asked the company a few years ago and got a reply that the membrane is actually OK up to 72 hours.

 

3.   The reason to use a u-bottomed plate in the first step for 28h and then transfer all to an ELISpot plate for the last 20h is to allow cells to stick all together, to facilitate interactions between the cells (in opposite to flat wells in ELISpot plate). 

 

4. Recently, we made a mistake performing ELISpot assay with MSIPN4550 plates instead of using MSIPS4510/4W10 plates as recommended by Mabtech (both from Millipore). According to the Millipore website, however, the difference between each plate type is only the sterility issue. Do you think it will make any difference in terms of spot development?

 

Kind Regards,

J

 

[Jens@mabtech.com]

Hi again J,

 

1. Yes, I’m referring mainly to monocytes in PBMCs (and the occasional circulating DC). To my knowledge, B cells don’t secrete IL-12. The comparison between frozen and fresh PBMCs can be done of course, but regardless of B cell- or DC-loss I think you will find more spots in the fresh sample since all cells, including monocytes, generally feel better. Speaking of which, do you check viability of your thawed PBMCs? With R848 I’m sure you will see IL-12 production, great that you’re willing to try it. In addition, I forgot to mention it in my previous response, but we do have a new human IL-12 system. It’s not a revolution, but the new antibody pair is slightly more sensitive compared to the old pair (see attached pdf with sample data). The new ELISpot-kit can be found here: https://www.mabtech.com/products/3455-4apw-2_human-il-12-p70-elispotplus-alp

 

2. It is definitely OK to leave cells on the plate longer than necessary, even if the analyte in question has fast kinetics. Our earlier comment about membrane being ok still after 72 hours still stands. Great that you’re willing to try out stimulating anti-CD3 (and possibly anti-CD28), that really should do the trick.

 

3. Although a fair argument, adding enough cells to a flat-bottomed well will give you satisfactory cell-cell interactions. So what is enough? Well, you have added 250,00 cells/well which is well enough. I can’t bring any firm data right here and now, but numbers down to 100,000 cells/well are in our hands generally good. Thus, you’re in the clear, no need to use u-bottom shaped pre-incubation steps. The thing you need to think about is that ELISpot, being an “accumulative” method, captures cytokines during the entire period of incubation. If you incubate your cells in the ELISpot plate for only 20h instead of the recommended 48h, you reduce the sensitivity of the assay since you don’t capture any of the analytes that were secreted during the first 28 hours. It doesn’t explain 0 spots for IL-12, but it certainly can be a part of the issue. Please use the R848 for IL-12 and the anti-CD3 (and possibly anti-CD28) for IL17A, IL-5 and Granzyme B and incubate in the ELISpot plate for 48h and I think you will get much better results.

 

4. The non-sterile MSIPN4550 is not optimal, especially since you’re using quite long incubation times. But seeing as your problem has been lack of spots rather than strange looking results, I think you haven’t had any contamination problems yet.

 

Kind regards
Jens

Human IL-12 (p70) sample data.pdf

[Questions from customers]

Hi Jens,

 

We do check cell viability and adjust the numbers of living cells before growing them in the plate.

 

                The protocol we used 2 step-culture was modified to measure both granzyme-B and IL-12 in that experiment only. As for IL-17 experiments, we did try to incubate cells in ELISpot plate for 48 hrs before without success. To be on safe side, we probably will incubate cells with positive control/drug on the plate for 48 hrs for every cytokine from now on. We will let you know how the experiments turn out once we got the results.   

 

Kind regards,

J

 

[Jens@mabtech.com]

Hi J,

 

Yes, please keep us posted on the progress of your experiments!

 

Kind regards,
Jens