Sounds like your plate isn't being kept completely still during the incubation 

I agree. Which capturing antibody do you use? In many/most ELISPOT assays only secreted products of your cells (like immunoglobulins) are bound by the capturing antibody but not the cells itself. This is why you shouldn't move the plate while incubating with your cells. If this happened, many of the spots look more like shooting stars rather than real spots. I hope this helps!

Can you show the image? The spot you see by your naked eyes could be not the spot since real spots are very small. The black streaks you see could be dead cell/tissue cluster or dust. But better is uploading the pic, I can tell more.

My first thought when you said spleen cells was that the cell prep was too dirty to get a good spot development.  Or there are too many macrophages crawling on the pvdf, displaying a cytokine that's reactive in the assay?  That is said from the perspective of one who hasn't done ELIspot assays.

The only other thing I can think of, knowing the theory behind the ELIspot is that there is too much disturbance (shaking, movement, vibration) during development, especially during the cell incubation period.

Depending on the assay, substrate and incubation time of your substrate, you can definitely see also real spots with the naked eye. But I agree, a picture of your spots would definitely help.

I strongly agree with previous comments. should not move plate while incubating your cells. how long you are incubating cells? I think it is good to incubate ON in cell culture incubator without disturbing the plate. are you looking for secreted immunoglobulins or cytokines? I usually incubate B cells ON to identify constitutively secreted natural IgM and IgGs. It works well. 

Yes, an image would be great to give optimal feedback. Most likely you deal with cells having moved during incubation, as already suggested. This can happen if the incubator is opened and closed often during incubation, or the incubator is closely located to a centrifuge. Also, if the stimulus was very strong, cells might move across the plate. It is also possible that you have a fungal infection  ongoing. DNA precipitates also cause streaks. So, again, an image would be helpful to nail down the cause of what you are seeing.

Thanks, Everybody! As everybody commented that moving the plate could be the issue, may be when others opened the same incubator would have moved the plate.

This time I make sure it doesn't move and let us see! :)

The black streaks maybe false positives, so it is not a good idea to count them as spots. also while adding the cells use a separate tip each time. and once you keep it in the incubator do not move for 20 hours for interferon gamma or 2 days if you are doing other cytokines like IL-2, or IL-17 etc.  also the optimum cell count is 8 million/ml. so keep that in mind too.  Good Luck!

Try to Read your ELIspot plate in a ELIspot plate reader rather than a microscope!.

What is this ELISPOT for? Assuming it is IFN-gamma, ideally with Con A stimulation you should see a large number of dots almost to the point of not being able to distinguish the spots. If you are having problems with Con A as well then you may have to fix your splenocyte isolation. Also, as others have mentioned keep the plate stationary. Good luck!

Before elipsot , Do you purify T or B cells from all population of Spleen?..ELISPOT is working very well for sorting cells.

or  .. HOW many cells do you put in each well ..You should have  a monolayer of cells on the membrane.