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Green and blue background with TMB

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Hello Mabtech-team,


I purchased the product Cat#3845-2H Mouse IgM ELISpotBASIC (HRP). The problem is the background shows two colours, green and deep blue. Please check the attached image.


I used Protocol II, and the antigen is TNP-BSA. The cells are lymphocytes isolated from lymphnodes, 100 000 cells per well, without the step “Stimulation in vitro”. After adding Streptavidin-HRP, I incubated for 20min at 37℃. I used the TMB substrate Cat#3651-10.


I used PBS (pH 7.4) for washing, and the water used in the last step is ddH2O. In the picture, 7F and 8F are the same sample, the same reagents, the same steps – why is the background so different? And which one is correct? 


I have had similar experiences and results with other Mabtech ELISpot kits (different analytes, all used HRP). The green color is always in majority. I think that I followed the protocol carefully, but should I check the operation again? What step or what reagent preparation do you think is the key?


Some companies use the AEC coloring system. Is it the TMB system used here that is the key point? What is the different between AEC and TMB on a PVDF plate?


I should be obliged if you could help. 


Warmest regards!




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Hi L,


The color depends on what liquid you use to stop the enzymatic reaction in the last step. If you use water the end-result is greener, and if you use PBS the end-result gets more blueish. 


If you look at HRP-spots in our image gallery (https://www.mabtech.com/knowledge-center/image-gallery) you can see that for example Ferret IFNg ELISpot (HRP) is more blue (PBS in last step) and Human GM-CSF ELISpot (HRP) is more green (water in last step). 


I think what has happened to the plate is this: In a few wells (the blue ones), there has been some PBS from previous steps left in the bottom. Thus in 8F some PBS has remained in the bottom in the well when the substrate was added, whereas in 7F all PBS was successfully removed. The green color is in majority because in most wells you have succeeded in removing all PBS.


There is no harm done. You can trust your spot numbers as long as there is a contrast between spot and background. But for future assays, you could: 

(i) Stop the enzymatic reaction with PBS. All wells will be blue.

(ii) Really make sure that all PBS has been removed before adding TMB. Then wash with tap water to stop the enzymatic reaction. All wells will be green.

(iii) Use the same kit but with ALP instead of HRP. This phenomenon is related to HRP and never occurs with ALP detection. The product code for the corresponding ALP kit is 3845-2A (https://www.mabtech.com/products/3845-2a-0-mouse-igm-elispotbasic-alp).


A tip for knowing how thorough you should be when emptying the plate before adding the TMB substrate: Hit the plate against some paper towels repeatedly and quite hard until you basically can’t see any more liquid stains coming out on the paper towel. At approximately 1 minute into this video we show you how to do it:


Finally, the difference between TMB and AEC substrate is explained here:


As you can see, the AEC substrate gives a much more red result. I am however unsure whether the nuance of red changes with PBS and water. 


Kind regards,



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