Your reasoning makes perfect sense! Thanks for clearing up my doubts. Dr. Jens mentioned something about Mabtech not recommending the use of PBS-T, could you elaborate on that?
We are happy to help!
You are right, we do not recommend adding tween into wash buffer. The reason for this is simple: in our hands, with our protocol, it leads to darker ELISpot membranes. There is absolutely no benefit.
At the same time, other ELISpot practitioners are adamant on the use of Tween as a critical component in making ELISpot results look better. How can our viewpoints be so different? Well, it all comes back to how the protocol is setup. We always recommend that you should EtOH treat your plates prior to coating. This increases the binding capacity of the PVDF membrane, and combined with a good amount of capture antibody (1-1.5 ug/well), will always produce better results compared to not using EtOH pre-treatment. With our protocol, Tween provides no benefit and will actually "hurt" your results by making membranes noticeably darker after the plate has been developed.
However, in cases where you decide not to perform the EtOH pre-treatment, results will actually benefit from the use of Tween. Spots will appear more clear and the "dark-membrane effect" seises to exist, or atleast becomes much less noticeable.
I find the spots in the OVA well really strange too. I highly doubt the NHPs would have been exposed to it previously and could have antibodies against it. It must be some sort of over reactivity/unspecific binding. But then again I don't know. If subtracting the spots, should I only do so from the Ag-specific spots or from the total IgG spots too? It makes more sense to only subtract from the Ag-spec but I don't really have any experience.
For the blocking I also use cell culture media (R10: RPMI 1640 + 10% FBS + 1% L-G + 1% P/S) for 1 hour. Most papers I've read on B cell Elispots block for 2 hours with R10, do you think that can make a significant difference? In my first attempts at the the B cell ELISpot, I included a "No Ag" control well but was later recommended by a more experienced (adapted my protocol from them) group to include an irrelevant Ag instead. Do you think the "No Ag" control is better (I do remember having an average of 1-2 spots there)?
I agree, it is unlikely that NHP have been exposed to OVA, but what about their diet? Could there be eggs in there? One of my collegues pointed out that a certain percentage of Igs are very "sticky" and will attach to almost anything. This is especially true for IgM, but we occasionally see it for IgG as well in some human donors. We believe that the "No Ag" control is better and more relevant. If the OVA spots are due to specific IgGs that actually exist in these monkeys, the spots numbers should not be subtracted. In our minds the "No Ag" control is a relevant one that is enough for these experiments.
The blocking time of either 1h or 2h should not make any difference.
When you write "play with intensity parameter instead" do you suggest that I should add a "maximum intensity threshold"? I find that playing with the minimum only subtracts from my Ag-specific wells as their intensity is usually read as lower because of the high background tint in those wells compared to the other wells.
No, not maximum. I would increase the minimum intensity threshold to something like 50. What if you increase camera setting quite abit so that elispot images become much lighter, and then change count setting to:
Intensity minimum: 50
Size minimum: 20
Gradient minimum: 1
Algorithm C, emphasis BIG
That will maybe differentiate the dark antigen specific spots when you coat with antigen, compared to the really faint background spots in you OVA control.
By the way, have you seen our tutorial video on how to setup the AID reader?:
It might be helpful.
Another aspect we have not talked about is the possibility of changing your experimental setup where you label your antigen with biotin, instead of using it for coating. We describe it abit here: https://www.mabtech.com/knowledge-center/assay-principles/elispot-assay-principle/b-cell-elispot
In addition, you can read this paper from 2009 where it was first introduced: http://www.ncbi.nlm.nih.gov/pubmed/19696434
By reversing the system you consume much less antigen and the system many times becomes much more sensitive. It is easy to biotinylate antigens these days, many ready made kits are available.
Furthermore, we have just recently demonstrated in a JIM publication that the same approach can be done using FluoroSpot with several peptide labeled antigens at the same time. You are then able to look at cross-reactivity at the single cell level: http://www.ncbi.nlm.nih.gov/pubmed/26930550
It is the ultimate revenge of the nerds!
