Dear Janna,
Welcome to the forum. There are a number of possible reasons as to why you are getting these uneven membranes. Before I give you like 5 different reasons, could you maybe tell us a bit about your protocol? You have purchased an Mouse IgG ELISpot basic kit (3825-2H), but how about the plates? Which model of Millipore plate did you use and did you do the etoh pre-treatment prior to coating? Which substrate did you use and have you been using tween in your wash buffers?
Here are my speculations on what might be happening:
We see these sort of artefacts in relation to the usage of tween from time to time. Mabtech has never recommended tween since it gives no benefit in our pre-coated plates or if you do the etoh pretreatment prior to coating (which we always recommend). Tween makes the membrane more penetrable for detection reagents and it is easy to get variability in the concentration of tween in wash buffers. As a result, it is possible to get uneven penetration of detection antibody into the membrane and patches of darker areas are formed.
If you do the etoh pre-treatment it is important to get the membrane wet all around. Uneven etoh pre-treatment can result in something that looks like this. It is important to always prepared fresh etoh from a high concentration stock (preferably >99%). If the concentration of etoh is too low, the membrane cannot be activated properly, you get patches. Another thing to look out for when doing etoh pre-treatment is to never let the membrane dry out. I am doubtful, but you patches in the membrane could maybe be related to membrane drying up during the assay. Always keep it moist with plenty of PBS. Never leave it exposed to air for extended periods of time.
Finally, I have personal experience with something that look kind of like your artefacts. I had done the assay perfectly. I had developed my spots with substrate. I washed off the substrate with water. Went back to my bench and began to dry the plate in my hood. The plate was of the MAIPSWU10 model so after that the membranes had been drying for like 3-5 minutes I put the plate back into its under-tray. Water was still left in the under-tray so the membrane, in patches, were again exposed to water. I wiped off the water and dried the plate again. Despite being completely dry, the re-exposure to water, once the plate had began to dry, resulted in ugly membrane patches similar to what I am seeing in your results. It is a long shot, but maybe you recognize my story.
