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Poorly developed ELISpot membrane

Guest Lena

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Dear all, 

Recently my collegues and I are experiencing problems with our IFNg ELISpot assays as some wells do not develop properly (and stay white with some spots) - even tough all steps were performed with the correct volume of reagents etc. (this was checked multiple times by different people). I also found the entry of a similar problem in the forum, but our issue could not be resolved by applying the recommended tips. 

You find a picture of one of our plates attached (see e.g. well E7). 

We use MSIP plates and activate the membrane as recommended. For development, 100 µL/well of detection antibody are added for 2h and 100 µL/well SA-ALP is used for 1 h. Membrane development is usually stopped after 15-20 min. 

What could cause such 'blank' wells and how can we avoid it? 

Thank you very much in advance, 





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When you say "blank" wells It still looks to me like these wells contain spots. So all reagents have been added correctly, otherwise there would be zero spots. 

One feeling I get is that you use tween in your wash buffer? Right or not? 

Tween is something we do not recommend. It makes reagents penetrate deeper into the PVDF membrane and this is only beneficial if you do NOT etoh activate the membrane. Since you do perform the etoh activation (which is good, sensitivty of the assay increases) tween gives no benefit. However, it can lead to a purple haze in the membrane. Some wells get less purple haze than others and you end up with results like yours. 

Many times when we ask if people use tween in their wash buffer they say no. However, they use the same wash buffer as for ELISA, and here you actually have tween in there. So easy to miss. 


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  • 5 months later...
Guest Idania

Hi Christian,

We are experience a similar problem with the difference that we are using the IFNg coating plates from Mabtech, so we don't EtOH activate the membrane. We are testing human PBMCs response against peptides. Initially, we were using only PBS for washing as recommended, but, because the long term culture of the cells, we were having high background. Thus, to try to decrease the background, we added 5 wash with PBS-0.05% Tween after cell stimulation following by 5 wash with PBS and we continue using only PBS for the other washes. Most of the plates are developing well without the purple color; but sometime, some wells do not develop properly and get the purple haze that interfere with the spot development and counting. Please see below the picture of one of the last plates (please check row D), the purple haze happens only in some wells and not in all the plate. 

I really appreciate if you can give us some feedback on how we can resolve this issue or alternatives to reduce background when using long term cultured PBMC without PBS-Tween.

Thank you for your help,


I am having problems to attach the picture


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