We are developing an ELISPOT to measure antigen specific mouse B cells. We are using a standard protocol in which PVDF microplates are activated with 70ul ethanol then washed. Antigen solution is then added overnight. Wells that are not part of the experiment are either stoppered or given coating solution without antigen. We often notice that during this antigen coating stage the solution drips through the wells! This perfusion is not even over the plate wells and indeed some wells do not leak at all. Can anyone advice how to overcome this problem?
Try Millipore MAHAN4550 mixed cellulose plates. You can pre-wet with PBS only, but it isn't necessary.
Are you pre-wetting the whole plate or just the wells included in the experiment? Are you also noticing the leak in the experimental wells?. Which ever way the wells are not supposed to leak. Have you brought this to the attention of the manufacturer. You may have been supplied with a faulty batch of plates. You may also switch to the cellulose plates that do not require pre-wetting. Also becareful not to touch the membrane with the pipett tip, as this may damage the membrane.
MIchael, I am sure there is a good reason for you to use PVDF plates (which exhibit a different mechanism to bind protein compared to nitrocellulose plates). The two most common reason for leakage are:
1. Too high volume of Ethanol for prewetting. Please, use only 15 ul per well. Other wise the membrane is saturated, and Ethanol leaks into the underdrain, promoting further leakage throughout the assay.
2. Older format PVDF plates. New plates have an optimized distance between membrane and underdrain. Please use the MS plates (e.g. MSIPS4510 or similar). In case you are already using them I recommend to check with Millipore whether the lot you used has already this improvement.
Your plates shouldnt leak. I agree with the above suggestions - check you dont have a faultly batch and decrease the amount of ethanol you are using. Plus if you arent using these wells in your assay why do anything wth them? just leave them empty
We had the same problem, together with a very high background. We contacted Millipore and they didn't know how to solve the problem. Finally we compared with and without Ethanol pre-wetting, and without worked very nicely. We wash the plate once with PBS instead.
Good luck
I have been using the Millipore MAIPSWU10 plates in my ELISPOT assays and either pre-wetting with 50ul of 70% EtOH or 15ul of 35% EtOH, depending on the protocol. I then immediately wash out the plate with PBS several times. I always verify that during the pre-wetting step the wells are not leaking: as long as the membrane darkens evenly, I do not experience issues with that well. Have you verified that your leaky wells weren't leaky prior to antigen loading? Also, in the past, we did have some issues with evaporation from wells on the edge of the plate, so I often times will avoid using those wells. As mentioned above, if you are getting consistent leaking from all of your current plates, I would compare lots.
Best of luck!
You have to be careful that the bottom of the membranes do not touch anything. If it does touch something the retention of the membrane is compromised. Hope this helps.
Pre wetting the whole plate PVDF is better option to get good transfer than nitrocellolose so try it
Thank you all for your comments:
Donald and Felix,
Will try the cellulaose plates.As described we do make sure to wet or at least stopper all wells, including those not in the experiment.
Sylvia,
The plates are indeed MSIPS4510 and I'll check with the manufacturer.
Rebecca,
For the first adsorption step there is no need to use all the wells. But later on for washing etc if all the wells do not contain the same volumes (or arenot stoppered) the vaccuum doesn't work correctly.
John Paul,
Don't know whether the students first found the problem after placing the plate on paper or but the tip is a good one!
Paula, Sara, Doris and Shah
Thanks for the tips.
Will report back with progress,
Thanks again,
Michael
I've experienced various problems with ELISPOT, generally related with the background, but we've never found leaking plates. You might consider to add PBS to them, wait and see if they leak. Bye the way, we also find that it is best to skip the ethanol soaking step.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA