change of background color after development(B cell ELISpot)

[Guest Eunju Jung]

Hello
I am performing a B cell ELISpot assay to evaluate the immune response to influenza vaccine, and I am contacting you because the background color changes strangely after development.
I used your ELISpot plate ( code:3654-TP-10, batch: R1CB64891) and ELISpot kit (code:3850-2H).
As spots appeared during the development process, they were washed with deionized water and the plate was inverted to air dry. When freshly washed, the backgrounds were all light blue in color (as we had seen in previous experiments), but when we checked on Monday after a two-day weekend to allow for sufficient drying, some wells had yellowish backgrounds.
I'm attaching a photo of the plate and a photo of the spots counted in the ELISpot reader. As you can see, wells 4D through 10D, 4E through 10E, and 5B,6A,6B,8A have yellowish backgrounds. (For better understanding, the ELISpot was run with 8 samples from columns 3 to 10, where rows A,B are PC (2 replicates), C-E are vaccine antigen (3 replicates), and F-H are NC (3 replicates)).
I have performed ELISpot with the same protocol before (spot count after two weekends of drying), but this is the first time that the background color has changed.
I'm not sure if I can trust the results. I ask for advice on whether I should continue with the experiment.

[Christian@mabtech.com]

Hey, welcome to the forum!

The membrane and spot color are influenced by a number of factors. The substrate you use, how long you develop and how you turn off the substrate reaction (either tap water, DI water or PBS). For HRP and TMB we don't recommend tap water. Have you used tap water in this case in any way? The yellowing of the HRP spots can occur using tap water and we therefore recommend DI water or PBS. I would in your case actually try PBS since this pH controlled. 

In addition to this I know from experience that "drying behavior" can have a dramatic impact. 

With "drying behavior" I am talking about many things: drying speed, amount volume liquid left when drying begins, re-wetting during drying and relationship of liquid on front side and backside of membrane once drying commences. 

When I go to turn of the substrate reaction of my plates I try and do it in a very controlled manner. I throw out substrate and add liquid in all wells three times. Once done I remove underdrain wash the backside of the PVDF membrane but I quickly make sure I re-fill the plate with new PBS so that all wells are kept full of liquid. 

I then empty the entire plate at once and tap the plate against a tissue.  I carefully wipe off underside of PVDF membrane. I want all wells equally "emptied" and wiped on the underside. 

After this, the crucial part: airdrying the membrane

If the drying process begins but membrane in a few wells is re-wetted, you will get strange darkness of wetted wells compared to all other. Re-wetting can easily happen if droplets of liquid are laying around somewhere on the lab-bench: must be avoided and understand that backside of the plate is very vulnerable without the underdrain in place! 

Secondly, if the last decant of liquid is not even across all 96wells and the drying process begins with some wells having 6ul of liquid still in the well, but others have 3ul, that can have an impact since drying process will be fundamentally different in terms of time.  

The above information might apply to your case but it could be something else. But I think the above factors are involved. Nevertheless, despite this, you spots look really good. The spot-counts look accurate despite the differences in background and spot color.