Peptide length for ELISPOT?

[Guest Guest Andrew]

Dear all, I've recently performed my first few ELISPOTs. 

I'm checking for reactivity towards both CD4 & CD8 tumor neoantigens. I have been using synthetic 29-mers representing the mutant amino acid and 14 flanking residues on each side. I use either tumor-infiltrating lymphocytes (20,000/well) or lymphocytes from the tumor-draining lymph node (50,000/well) as effectors, and naive splenocytes as APCs (500,000/well).  

I noticed that the number of spots above background (medium only; just T cells + splenocytes) I get is variable and often times quite low; generally between 5-50 spots above background per antigen. For Concanavalin A stimulation I get about 500-1000 spots per well, and for the positive control antigen I get 10-50 spots above background. I notice that often papers report spot counts in the 100s for antigens. I also notice that almost every other paper I've encountered utilizes 8 to 10-mer peptides for CD8 stimulation, and 15-mer peptides for CD4. 

I was wondering if I could also expect a significant increase in spot counts from switching from 29-mer peptides for both CD8 and CD4 stim to using the minimal MHC-I epitope (8 to 10-mer) for CD8 stim and the shorter 15-mer peptides for CD4 stim? 

[Guest Guest Andrew]

Sorry, unrelated but I'm also wondering if it is advised to lyse the RBCs in the splenocytes before utilization as APCs? 

[Christian@mabtech.com]

Hello Andrew,

When you add 29-mers these need processing and presentation by APCs. If you on the other hand add 8-10mers and 15mers these can be presented by many more cells per well. Not only the APCs. This way of doing is the standard way and I certainly think this is advisable. 

In general, adding 500k splenocytes to an ELISpot well can result in IFNg backgrounds. There are many T-cells and NK-cells among those 500K cells that cannot act as APCs. What if you only add the tumor infiltrating lymphocytes, how many spots per well do you get then? Furthermore, If you as a control only add the 500k splenocytes into one well, how many spots do you get then? 

Would be good to get a sense for how many spots/well you have in background.

Removing red blood cells is something many people do. A lot of RBC can lead to strange membrane staining, but one would be surprised how much red blood cells is ok to add without causing issues. The lysis can be rather harsh treatment which can impede any cellular function. 

[Guest Andrew]

Christian, thank you so much - this is extraordinarily valuable insight! I will implement all of those modifications (peptide length changes, splenocyte only and T cell only controls) for our next round of ELISPOT. If I would like to remove RBC, but the lysis might not be recommended, is there a particular way you would recommend RBC removal? 

[Christian@mabtech.com]

Happy to help!

Either you remove the RBC or you don't. I personally tend to avoid it because its such a rough treatment, especially since mouse cells are kind of sensitive (compared to human cells). There are these RBC lysis buffer solutions out there all based on ammonium chloride. Any provider will do I guess.

In my own research with human granulocytes I stopped doing RBC lysis and the quality of the spots went up quite a bit. I was then surprised that a fair bit amount of RBC did no really impede the ELISpot assay. Sure, if you have massive number of RBC in your cells they will cause problems, but cell pellet can be rather reddish and still not stop ELISpot from working well. 

Consequently, if possible, I like to avoid doing the RBC lysis.