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B cell ELISPOT, blurry spot

Guest Eunju, Jung

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Guest Eunju, Jung

Dear, Mabtech team


I am measuring influenza antigen-specific antibody secreting cell using Human IgG ELISpot.

 But I noticed that spots were not clear and some spots were shown as a clump.

I am using cryopreserved PBMC and when thawing PBMC, I use DNase to prevent cell clumping.

Is there anything I can do to improve blurred spots? Or will it go well, if I try increasing a concentrate of DNase?


In attached ppt file, first slide is a result of spots development during 15 minutes.

As 15 minutes, background was too darken to distinguish spots. So I shortened the development time. 

Next slide is results of two developments during 3min, 30sec and 1min,45sec.

Please give me a help with this spot problem.



 Eunju, Jung

B cell ELISPOT.pptx

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Dear Eunju,

In my opinion you spots look pretty decent. In addition, you post very nicely demonstrate the problem with overdevelopment with substrate in ELISpot. Go for too long and the contrast between spots and membrane goes down to the point were readers have difficulties picking up the spots correctly. 

Now, there is one thing I recommend for making this situation better: You can try and see if you can switch to a reversed B-cell ELISpot instead. 

In the revered B-cell ELISpot you use an anti-IgG antibody in the bottom and capture all IgGs secreted by the added B-cells. Then in the detection step you add the biotinylated antigen and finally visualize spots using streptavidin-ALP. The benefit is that you consume very little antigen this way and the quality of spots often go up. Many times you get an increase in spot numbers as well (i.e better sensitivity). I have attached a PPT with a printscreen from one of our brochures. It shows how the reversed system have much cleaner membranes compared to coated antigen. 


In order to do your assay in the reversed way you can buy biotinylation kits from companies such as Innovabiosciences. Very easy to use. 






Example of clean membrane using Reversed B-cell elispot.pptx

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