I've been trying to detect IL-12p70 from supernatants from PBMCs, however I keep getting negative absorbance values. I've been using the DuoSet from R&D. But, I was wondering if anyone else had this issue and if they have been more successful with a different kit?
What are you stimulating your cells with? I have had similar issues with this human kit from R&D. Have you tried CpG?
I've been stimulating the cells with various TLR ligands and also I've been infecting the cells with viruses and viral components, depending on the specific experiment. I haven't tried CpG. I've been using Streptococcus pneumoniae as a positive control (and I've ordered Poly IC for a range of experiments I plan to carry out soon). I've never managed to get positive values (not even my positive control). Yet, I've been detecting many other interleukins using the same samples, so I know the stimulation/infections have been working to some degree. I know that eBioscience also have a IL-12p70 kit. Do you have any experience with that kit?
I haven't used the eBioscience one so I'm not sure what it's like. I've heard that IL12p70 can be very tricky to pick up. Have you done any time course experiments to see if you can pick up the IL-12 mRNA (if you have the primers)? Might be worthwhile trying before purchasing a new kit which could have very similar issues.
I've just recently purchased a PCR machine so I think that will be my next step. Thanks for your help!
I am not expert with IL-12 p70 in particular but culture time is critical for a proper harvest of certain cytokines. IL-12 is produced after relatively quick incubation times, so you should check on that. Here is a paper that might help you, although I suggest you read further.
Good luck
In my past experiment, I used mouse bone marrow-derived pDC to do work similar to you.
When the DC incubated with CpG(1ug/mL) or TLR2 agonist for 24hr, we usually got positive results by eBioscience ELISA kit. And the concentration of IL-12p70 is around 100pg/mL when DC culture supernatant was used as test sample.
Hope this information helps.
Li Sheng Chang - Thanks, I think I'll try the eBioscience kit then. I'm also incubating for 24 hours, so hopefully, I will also be able to get some results from it. Thanks!
Paula Maguire,
I need to correct my last answer in regard of concentration of IL-12p70.
The concentration of IL-12p70 in pDC culture supernatant following incubation with CpG for 24 hours is around 1000 pg/mL. The cell density is 200000 cells/well seeded in flat 96-well culture plate.
Best,
Try this ($240)
http://www.novateinbio.com/en/human-elisa-kits/76331-human-il-12p70-elisa-kit.html
Paula,
I was just wondering if you had any luck with picking up IL12p70 in the end? Having the same issues as before with the R&D ELISA kit and so has a colleague in our department. If you could shed any light on this it would be much appreciated.
Best wishes,
Aoife,
I ordered an eBioscience IL-12p70 kit. I've literally just analysed the results from my first round of ELISAs using it and it's picking some up! I've so far analysed three different stimulations and in two of them I was able to detect IL-12p70, whereas I never was able to detect any IL-12p70 with the R&D kit (and I tried many, many different stimulations). I've attached the link to the kit I bought. It's a good bit more expensive than the R&D. But, if it continues to detect IL-12p70 for me, then it's definitely worth it.
Hope this helps.
Best wishes,
Paula
http://www.ebioscience.com/human-il-12-ready-set-go-elisa.htm
Hi Paula,
Thanks for this- It does seem to be an issue with the R&D kits...Glad to hear you're picking some up with the new kit! I'll look into the eBioscience one myself.
Best wishes,
Aoife
Hi Aoife,
Hopefully, you have some luck with it. I've also incubated the samples overnight on the eBioscience kit to maximise the sensitivity. It's still a little tricky, but so far, I've had a lot more luck with it that with the R&D kit.
Paula
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