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Uneven background in mouse IgG ELISpot

Guest Janna

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I have been working with the mouse IgG elispot kit (product code 3825-H) to detect both total IgG and ag-specific IgG to a virus-like particle (VLP) vaccine candidate in mouse splenocytes. For the antigen-specific, I coat my plates with 5ug/ml of the VLP. In both protocols, I am seeing a very uneven background signal both within and between wells. This problem is more pronounced with the ag-specific assay, but is also present with the total IgG assay. I attached a picture of the dry plate. As you can see, in some wells there is a stripe of dark purple background, with no background in the rest of the well. In other wells, there is no background at all.  Do you have any ideas why this might be happening?

Thank you!





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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.  

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Hi Janna,

In connection to Christian's speculation in the last paragraph, re-exposure to water, I'd like to add that maybe this is not due to the specific re-exposure to water but just simply insufficient emptying of water from the plate. Perhaps there is still water droplets left on the membrane when you leave them to dry? At 1 minute into the following video, Christian shows a good way of emptying liquid from a plate (i.e. tap the plate against paper towels and judge the "emptying efficacy" by looking at the amount of discarded liquid visible on the paper): 


The video above concerns a certain fluorescence enhancer-reagent, but the same principle adheres to any liquid that is to be emptied from an ELISpot or FluoroSpot plate.

Please also make sure to dry the plates upside-down to reduce the risk of remaining water droplets interfering with the drying process. The following image shows how we usually dry plates in-house:


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Thank you all for your quick replies!

Christian, to answer your questions:

I was using the MAIPSWU10 plates, and I think what you suggested about re-wetting may have something to do with the issue. I always tap my plates dry as in the video above, but there are sometimes still drops of water in the trays. I will be starting another round of assays next week, and I ordered the MSIPS4W10 plates to see if that makes a difference. I was not aware that the wells were so sensitive to re-wetting during the drying stage.

I did pre-treat with EToH, but I did not make a new solution each time, so that may also be part of the problem. After this step though, I was very careful to not let the wells dry. 

My wash buffer does not include tween. I simply used filtered 1x PBS. 

The substrate I used was the TMB for substrate systems from Sigma (http://www.sigmaaldrich.com/catalog/product/sigma/t0565?lang=en&region=CA). 


Thank you for the video and the tips. As I mentioned above, I do tap my wells on paper towel to dry them. I was not aware that plates should be left to dry upside down though. I will definitely try that next time. 


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Hey again Janna,

Good that you recognized the story about rewetting. Also drying the plate in the upright position is not advisable. Could be the culprit. 

I actually like the MAIPSWU10 plate quite a lot since it is so reliable. You never get leakage as long as you control so that underside never gets touched by any drops of liquid. We talk about emptying 5:25 in the youtube video below. You need to keep the plate and undertray as one during emptying, otherwise you risk getting drops on the backside and thereby inducing leakage. 

But trying the MSIP model is a good option as well. You can keep the underdrain on the that plate throughout the assay, atleast that is what I do. 

You should buy your substrate from Mabtech, not from Sigma! :) 

We have TMB substrate right here: https://www.mabtech.com/products/tmb-substrate-elispot_3651-10

Specific for ELISpot! The sigma substrate could be suboptimal. 


Here is the tip (5:35i into the video) on how to handle washing MAIPSWU10 in order to avoid drops on the backside:


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