Maximum sensitivity in ELISpot?

[Questions from customers]

Hi Mabtech,

In terms of sensitivity, I see that the ELISpot assay can detect as little as one spot. In your experience, if there was only one antigen reactive T cell in 600,000 PMBC plated and you pulsed the well with presented antigen (for that one T cell), would that T cell secrete enough IFNg over time in order to be detected in this system?
 
I am looking for a needle in a haystack, and I want to make sure this assay is an appropriate one. If it isn’t, can you recommend another product that would be sufficient?

Regards,
M
 

[Jens@mabtech.com]

Hi M,

The maximum sensitivity of ELISpot is hard to say, because in theory it’s unlimited. Let’s say you have an ELISpot well big as a bucket, and you add 100 billion cells in there, and add 1 cell you know are responding to your antigen, then yes, you would theoretically actually be able to detect it. It is a bit dependent on the cytokine secretion over time, as you point out, and that in turn depends on the particular cell and antigen. Unfortunately, we can’t say for sure exactly how much cytokine must be produced to see a spot. But normally, for low-frequent antigen-specific T cell IFNg-responses, many papers show a handful of antigen-specific spots in a population of several hundred thousand PBMCs.

Of interest, we have a customer that FACS-sorted exactly 1 cell into an IFN-gamma/IL-8 FluoroSpot plate well: https://www.ncbi.nlm.nih.gov/pubmed/28754679

The authors of the linked paper were by that procedure able to first confirm that recent thymic emigrants secrete only IL-8 and not IFN-gamma, and then – by removing the still alive cell with a pipette and transferring it to another plate for cloning – show that the very same cell transitioned into an IFN-gamma producing Th1 cells. That’s not 1 cell in a population of 600 000 cells, which is what you are asking for, but at least it shows that it is possible to detect one single responding cell using FluoroSpot (and FluoroSpot and ELISpot have similar sensitivity.)

ELISpot and FluoroSpot are the most sensitive assays out there, so if you are looking for a needle in a haystack, then these assays are the way to go. 

Kind regards,
Jens