Human pan IgG ELISpot - dark membrane and white spots

[Guest Emily]

Hi there, I have had some issues with a recent set of ELISpots I ran on human PBMCs and am hoping for some assistance. I followed the Mabtech protocol's recommendation for PBMC stimulation and used 10ng/mL IL-2 and 1µg/mL R848, with a completely unstimulated control as well. My DMSO concentration in my culture was 0.1%. I plated my cells after the stimulation. 

I ran an IgG4 ELISpot and an IgG ELISpot, following instructions for total IgG detection rather than antigen-specific IgG detection. I pre-activated both plates for both ELISpots in the same way, with 35% ethanol for about 1 minute. I then plated 100k cells for my IgG4 ELISpot and plated both 50k and 100k cells in separate wells for each condition of my IgG (since this was my first run, I tried both conditions). My IgG4 ELISpot was successful, and I was able to detect spots following the IL-2/R848 stimulation but not in the unstimulated control, and the membrane remained light coloured. However, for my IgG ELISpot, the membranes of my wells were stained very dark blue and oddly, there were tiny white spots throughout the well. Surprisingly, my unstimulated control well had many more white spots than my IL-2/R848 stimulated well. The wells with 50k cells vs 100k cells both had this issue.

Could you please advise me on what to try differently for a repeat study? Thank you!

 

IgG 1 = unstimulated IgG 1.heic

IgG 12 and IgG4 12 = 10 ng/mL IL-2 and 1 µg/mL R848 IgG 12.heicIgG4 12.heic

[Guest Emily]

I should also add that I used PBS for all my wash steps and when I plated my cells in my ELISpot plate (post-stimulation), they are resuspended in PBS. 

[Christian@mabtech.com]

So I have some questions and comments:

1. You took isolated PBMC and pre-incubated them for 3 days with R848+IL2. You also had a parallell culture without any stimulation at all. At what concentration did you culture these cells? Did you check for viability prior to setting up these cultures? Why does these cultures contain any DMSO at all? 

2. After three days of culture you transferred to the cells to prepared ELISpot plates. Did you again check for viability at this stage? Did you wash the cells extensively prior to seeding them in the plate? This is very important because there is so much IgG produced during the culture. These will black out the ELISpot membrane unless you wash them away really carefully. 

3. Looking at your result the membranes look very dark. Either from excessive substrate development time or from too much Ig in the cell supernatant. 

4. When you have massive IgG bind to the bottom of the plate and you over-develop with substrate you can get these white dots in the membrane. My guess is that this could simply be from heavy units of precipiate "breaking" off the membrane, but that is a speculation.

Overall it would be interesting to know the viability of your cells. If one has too low viability there wont be any B-cells capable of secreting IgG. 

The IgG4 total result do look like real spots but they are very faint, indicating that cells are not feeling its best. Spots should look something like this: