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IFNg ELISPOT: Inhibition of background spots in wells for which the donor is seronegative

Guest José Manuel Marín

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Guest José Manuel Marín

Dear all,

I perform IFNg ELISpot with the mabtech reagents following the mabtech protocol and it works great.

I have realized there is a small fraction of donors which have a high background in the negative control which prevents showing any significant differences  between the negative control and the peptide stimulated wells.
Very interestingly, in those donors there are some other viral peptides (presumably the ones the donors have never been in contact with) that are much lower, or where the spot count drops to 0. In other donors that same peptide stimulates T cells properly, therefore it works perfectly.

Do you have any potential explanation for this?? This actually speaks for it that there should be a negative control peptide to prevent this "unspecific"/ unwanted activation. I guess it would also make sense to potentially use that "cleaned" background stimulation as a proper negative control to compare it with the other peptides for which one actually clearly sees additional spots.

Could anyone comment on this?

Thanks a lot.


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Dear Josema,

I have only experienced this phenomenon in rare instances when others have shared data with me. But it would seem that some peptides, just like you point out, can magically make unstimulated IFNg spots go down in ELISpot assays. 

Since we are dealing with a living cell culture, the phenomenon can be elusive to understand since it potentially could involve a "bystander effect". For example:

- A particular peptide is contaminated with a TLR ligand that sets off a special subset of monocytes that start secreting IL-10. The response will vary in donor to donor depending on the size of monocyte subset.

- The secreted IL-10 binds to the T-cells responsible for the background and downregulate their secretion before they are able to start releasing IFNg during the ELISpot. 


Another more sinister possability is that the peptides causing the reduction in background spots are somehow killing the T-cells in the ELISpot assay. 


If in fact it existed a special peptide that turned down unstimulated IFNg release without being contaminated with anything or due to causing apopotosis, that would be valuable research tool. 



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