IFN-Gamma ELISPOT No Development?

[Guest Eva]

Hello,

I'm having a problem with the IFN-gamma ELISPOT assay (3420-2A). 

We've been using an optimized version of the protocol that you recommend since years ago our collaborators optimized it to limit the high background that they were getting with PBMCs.

I've run about 200 of these plates (Millipore  MSIPS4W10) and I've found that about 10% of the time, the plates do not develop properly. After the color development step using Biorad's AP Color Development kit (https://www.bio-rad.com/en-us/sku/1706432-ap-conjugate-substrate-kit?ID=1706432), I get white wells. My troubleshooting has been to soak these plates in 1x PBS for 1-2 hours, then redevelop them starting with the anti-IFN gamma biotinylated Ab from your kit. I tried doing that yesterday but when the color development step was complete, my wells were completely "purpled-out." The strange thing about this time was that I ran another 3 plates at the same time, batched, with the same coating antibody, the same biotinylated antibody and a different streptavidin batch number, and they came out perfectly.

Our protocol is as follows:

1. Pre-coat plates with 5ul of anti-IFN gamma coating Ab in 10mls of PBS per plate, 100ul per well (we do not activate our plates with 35% EtOH)

2. The next day flick out the coating Ab and wash 6x with 200ul 1x PBS. Then plate 100,000-200,000 recently thawed cryoperserved PBMCs into each well of each plate. 90ul of PBMCS and RPMI and 10% FBS media per well. The cells are thawed the same day. Then stimulate the wells with 10ul of peptide pools for a final concentration of 1ug/peptide/pool/well/ml. Positive control is PHA, negative control is 10ul of 10% DMSO in PBS which ends up with a final concentration of less than 1% DMSO per well, since that is what our peptides are reconstituted in. Once peptides are added, fill up wells to 200ul and put in the CO2 incubator at 37degrees for about 17 hours.

3. The next day, flick out cells and wash each plate 6x with 200ul of 1x PBS. Then apply 100ul per well of the anti-IFN gamma biotinylated Ab from the kit at 5ul per 10mls of PBS per plate. Incubate plate at room temperature for 1 hour. 

4. Flick out biotinylated Ab and wash each plate 6x with 200ul of 1x PBS. Then apply 100ul per well of the streptavidin from the kit at 5ul per 10mls of PBS per plate. Incubate at room temperature in the dark for 1 hour.

5. Prepare the color development solution. 400ul of the buffer with 100ul of reagent A and reagent B in 10.6mls of DI H20 per plate. Flick out streptavidin and wash each plate 6x with 200ul of 1x PBS. Then apply 100ul per well of the color development solution and watch it develop for 15 min.

6. At 15 min, flick out the color development solution and apply 100ul of PBS with .05% Tween. Wait for 10min.

7. Flick out the PBS-T and wash the plate 4x in tap water. Leave plates to dry overnight before imaging.

 

thank you!

Best,

Eva

[Guest Photo followup]

Here's a picture of a plate from the last time this happened (not yesterday's plates, those are still sitting in 1X PBS in case I can rescue them).

Thanks!
Best,

Eva

[Christian@mabtech.com]

A few comments/suggestions from my side:

- In the 3 plates you have uploaded I do not see any positive control wells full of spots. Is this just a co-incidence? Or is it always like that? In general, when using a positive control like anti-Cd3 or PHA you get above 500-1000 spots/well atleast at 100,000 cells/well. It is good always having a few positive control wells in each plate to assure that cells and the assay is working well. 

- When I coat ELISpot plates myself I always do EtOH activation prior to adding capture antibody. I then use the recommended concentration of 1,5ug/well. In you example you say you add 5microliters of antibody to 10ml of PBS and then coat with 100ul of that solution. I would use 150ul as a comparison. You essentially use 0,05ug/well of capture antibody, which is too low. You could go down to 0,5ug/well but you would then need to add 50 microliters of mab to 10ml PBS when preparing your stock. This could explain a total lack of spots in general. 

- I think it would be good if you tested pre-coated plates in parallell to just get a feeling for how it could look when optimally prepared. Pre-coated is more expensive but it ensures an optimal result. You spend a tremendous amount of money and energy on generating this type of data so for me it makes little sense saving a few bucks on the final read out. Our ELISpot PRO kits come with one-step detection where 7b6 is directly conjugated to ALP:

https://www.mabtech.com/products/human-ifn-gamma-elispot-pro-kit-alp_3420-2apt-10

In this kit we also include our own BCIP/NBT Plus substrate. I have never seen the BIORAD substrate before. A substrate not optimized for ELISpot can have devastating effects. Is this BIORAD substrate actually a precipitating substrate? It needs to be otherwise you wont get any spots. 

 

 

[Guest Eva]

hi Christian

Thanks for getting back to me.

Our positive control was PHA and usually we get nice development where we see easily countable spots and our PHA wells are blown out from so much expression of IFN-gamma due to activation. We always have a positive control. The problem in this case was that non of the controls showed up because the entire plate was stained purple.

I've run about 200 ELISPOT plates using this exact protocol every time and my lab has run at least 300 with this protocol. We've never had a problem like this. We have spent months optimizing this assay (with and without pre-coating, with and without EtOH, different titrations of Ab to use) and this is the combination that works the best for us--we are looking at IFN-gamma expression in PBMCs from people living with HIV who are ARV-suppressed's response to HIV peptides. Any other combinations have yielded too much background. I also ran this set of plates batched with another set and the other set developed perfectly. 

We've also used the biorad substrate for years and have never had a problem. As far as I know, it is a precipitating substrate.

I've attached pictures of one of the plates that we ran batched that did develop. Thanks!

[Guest Eva]

also, the PHA is well H12. thanks!

[Christian@mabtech.com]

Sorry for not getting back earlier Eva. I am out traveling but have looked at your response from time to time and here is what I have to say:

- You certainly have spots in the last uploaded elispot images. I dont have any magical recommendations for you that can explain your sudden lack of spots in some plates. But If I were to speculate it would be that the cells are not in good condition. I say this because if it was an issue with reagents not being added correctly, the first uploaded plates would be completely blank, but they are not. If I look carefully I can see wells that seem to have real spots in them. 

- So one scenario could be that there has been a mistake in dilution for example. Instead of adding 100,000 cells/well, only 10,000 PBMCs are added. They result is no response and PHA postivie control can suffer a lot as well due to it needing a certain level of cell to cell contact to work well. 

- Another thing could be viability of the cells. If thawed PBMC are handled improperly, for example they are left for long periods in freeze medium before being transferred and washed by cell culture medium, quality of the cells can go down fast. Say for example you thaw 10 tubes of cells from one donor simultansouly. Tube 1 only gets 1min of freeze medium treatment after being thawed in a water batch,  but tube 10 will get 8min. That can have a big impact. 

[Guest Cyril]

Eva did you figured out the origin of the huge purple/dark back ground that you had in your wells?