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No spots for IFN-gamma

Guest Kenneth2020-09-15T11:46:06Z

Hi, I am getting zero spots and low background in the human IFNG elispot assay. I am just trying out elispot assay before moving to other applications with human IFN-gamma protein. So I am using 293F (suspension type 293 derivative, serum free) and transfect a CMV driven flag tagged IFNG from geneEZ ORF plasmid, co-transfect with GFP reporter.

https://www.genscript.com/gene/homo-sapiens/3458/ifng.html#NM_000619.3

I am using human elispot basic kit with all condition same as suggested protocol on website.

But there is no spot for many times i tried, with different cell lines. The reagents are new so I did not add IFNG protein as positive control. We don't have PBMC in the lab.

There is zero background, which is quite white.

Guest Kenneth2020-09-15T11:48:30Z

Also, I wish to ask does flag tag mask the monoclonal antibody? And moreover i want to know how your plus and pro plates works. Is it shipped dry for the membrane and pre-coated? So do I need to use ethanol to activate it before I put my cells in for incubation?

Christian@mabtech.com2020-09-16T13:50:34Z

Admin

Dear Kenneth, in all liklihood your transfected cells are not secreting the IFNg cytokine.

How many cells are you incubating per well?

We occasionally run transfected cells in ELISpot when looking at exotic animal IFNg like Salmon for example. We get massive amount of secretion so we have to keep incubation very short. Only about 2-4h. Rather few cells per well also, around 500-1000. Otherwise the whole membrane is full of spots.

Christian@mabtech.com2020-09-16T14:02:19Z

Admin

Hold on, you are running an ELISpot basic kit! What substrate are you using?

We have had experience before with people getting zero spots after trying to develop them using ELISA substrate. Will NOT work.

The substrate need to be "precipitating" substrate, like BCIP/NBT Plus:

https://www.mabtech.com/products/bcip-nbt-plus-substrate-elispot_3650-10

Guest Kenneth2020-09-17T02:41:44Z

I see! Thats why I am confused as both of them are TMB substrates. I think that is the problem. I had one time had some yellow spots on the membrane with TMB elisa substrate after drying. Thank you! I will try again and update you after acquiring the correct substrate.

The cells were seeded such that 1000, 100, 10 and 1 spot is expected, so with 10% transfection efficiency i seed from 10000 to 10 cells.

Christian@mabtech.com2020-09-17T07:56:33Z

Admin

Ah, very good that we found the issue! Mabtech TMB substrate for ELISpot can be found here:

https://www.mabtech.com/products/tmb-substrate-elispot_3651-10

Your strategy for the number of cells to incubate per well sound very reasonable.