Questions from customers Posted December 12, 2016 Report Share Posted December 12, 2016 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 Link to comment Share on other sites More sharing options...
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