High background

[tylsan]

Hi Mabtech!

I used your human IgG/IgA/IgM FluoroSpot kit (FS-050617) to look at total and vaccine specific plasmablasts after vaccination with different licensed vaccines. Overall the kit and protocol was well constructed and easy to follow! I used cryopreserved cells that I rested for 1 hour before moving them to the plates for incubation. The plates were coated with either the anti-IgG/A/M included in the kit or 10 µg/ml of inactivated whole virus (specific to the vaccine). The antigen specific wells didn't seem to work at all, but the highest concentration of cells worked well with the total IgG/A/M and are similar to our FACs data from these time points. However in certain wells there is really high background. I used a 12-channel multichannel pipette so I would expect that all the wells in the row to look somewhat similar, but they don't seem to be that way. Any ideas why this could be?

Thanks!

Fluorospot IgG:IgA:IgM.pdf

[Christian@mabtech.com]

Hey Tylsan,

The images of the fluorospot wells are bit low-rez but to me it looks like they are rather nice looking in the positive control wells. However, I am not following you completely about the experimental setup of the plate. In the attachment there seems to be duplicates for day 0, day 7, day 14. It then starts over in wells A7 and A8 (Day 0), A9 and A10 (Day 7), A11 and A12 (day 14). Is this a second donor? I dont understand how you used that 12 channel multipipette if you in fact had 2 donors. But even if you had just one donor,  I still dont really understand  

When you thaw the cells and let them rest for 1h, do you wash the cells before adding them to the FluoroSPot plate? If not, there is potential for build up of immunoglobulins in the cell culture medium during that 1h which will then generate some background in the membrane.

A lack of antigen specific spots, was that a dissapointment? Were you expecting many? One reason could ofcourse be that the frequency of those antigen specific B-cells are rather low. On the other hand another reason could be that the antigen is not suited for coating. Have you considered trying a reversed B-cell FluoroSpot like done in this paper: https://www.ncbi.nlm.nih.gov/pubmed/26930550

Another aspect. You total control wells are not packed with spots. In PBMC only around 10% are B-cells. Could you go up in cell number or even better could you enrich for your B-cells in isoaltion (https://www.stemcell.com/rosettesep-human-b-cell-enrichment-cocktail.html)? Furthermore, have you tried analyzing these donors fresh without freezing of the cells? Less trauma better response?

You are coating these plates yourself. Are you using the Etoh pre-treatment? For both controls and antigen coated wells?

 

 

 

[tylsan]

Hi Christian!

Thanks for the speedy resonse!

10 hours ago, Christian@mabtech.com said:

The images of the fluorospot wells are bit low-rez but to me it looks like they are rather nice looking in the positive control wells.

Thanks! I was happy to see at least part of it worked   Sorry about the low resolution. This is just the screen capture from the AID software.

10 hours ago, Christian@mabtech.com said:

In the attachment there seems to be duplicates for day 0, day 7, day 14. It then starts over in wells A7 and A8 (Day 0), A9 and A10 (Day 7), A11 and A12 (day 14). Is this a second donor? I dont understand how you used that 12 channel multipipette if you in fact had 2 donors. But even if you had just one donor,  I still dont really understand  

Sorry I realize I could have been clearer there. They are two different donors. I used a separate 96 well plate to make the dilution series for the samples so that I could transfer over them over easily. 

10 hours ago, Christian@mabtech.com said:

When you thaw the cells and let them rest for 1h, do you wash the cells before adding them to the FluoroSPot plate? If not, there is potential for build up of immunoglobulins in the cell culture medium during that 1h which will then generate some background in the membrane.

Yes after resting 1h, the cells were washed and resuspended and then transferred to my dilution plate before plating on the fluorospot plate.

10 hours ago, Christian@mabtech.com said:

A lack of antigen specific spots, was that a dissapointment? Were you expecting many? One reason could ofcourse be that the frequency of those antigen specific B-cells are rather low. On the other hand another reason could be that the antigen is not suited for coating. Have you considered trying a reversed B-cell FluoroSpot like done in this paper: https://www.ncbi.nlm.nih.gov/pubmed/26930550

The lack of Ag specific spots was disappointing. We see a significant increase in plasmablasts after vaccination with flow cytometry on the fresh cells and assume they are vaccine specific so we do expect to see some. The antigen is from the vaccine. It's a live attenuated virus that was grown and inactivated using Triton X-100 (0.5%), KCl (0.6M) buffer and heat inactivation. So the whole virus is still present and we hoped it would work for coating. Could a whole virus particle be too large to bind?

I talked with Peter at the SSI meeting last fall (if I remember correctly) and discussed doing a reverse FluoroSpot, but were unsure how to go about tagging our antigen since the antigen is a whole virus. Any ideas on tagging viral particles?

11 hours ago, Christian@mabtech.com said:

Another aspect. You total control wells are not packed with spots. In PBMC only around 10% are B-cells. Could you go up in cell number or even better could you enrich for your B-cells in isoaltion (https://www.stemcell.com/rosettesep-human-b-cell-enrichment-cocktail.html)?

Since we (right now) only have frozen cells we could enrich them using this? https://www.stemcell.com/products/easysep-human-pan-b-cell-enrichment-kit.html

11 hours ago, Christian@mabtech.com said:

Furthermore, have you tried analyzing these donors fresh without freezing of the cells? Less trauma better response?

We were hoping to do these experiments on fresh cells, but the collection started before we had unfortunately fully optimized this assay. I also have an FluroSpot incubating now from the same donors, but instead I stimulated these cells for 48 hours. We'll see how that turns out this afternoon. 

11 hours ago, Christian@mabtech.com said:

You are coating these plates yourself. Are you using the Etoh pre-treatment? For both controls and antigen coated wells?

Yes the whole plate received 35% Ethanol pre-treatment for approx. 30 seconds.

 

Thanks so much for your help!

/Tyler

[Christian@mabtech.com]

Here is some feedback:

Instead of doing a complicated enrichment of B-cells using beads I think you could start by just going up in cells/well to 400K. 

But I also taked to some people here at Mabtech and it turns out that not all Vaccines induce massive amounts of plasmablasts. We tested this a few years back with Pandemrix and here the cells really went crazy activated and doing an ELISpot/FluoroSpot without any pre-activation period of 3 days worked great. However, with other types of vaccines the response is not as strong. Please have a look in this paper:

https://www.ncbi.nlm.nih.gov/pubmed/?term=optimization+Ahlborg

 

In it there is a part very they evaluate some vaccines and not strong responses are seen directly ex vivo. 

So maybe in parrallell you should set up another FluoroSpot plate were you prior to the assay do in fact perform the 3 days activation with R848+IL-2. 

Doing a biotinylation of whole virus particles work fine. Here is kit that I think should work well:

https://www.expedeon.com/products/applications/immunoprecipitaion/lightning-link-rapid-biotin/

With it you can start by doing 1-color testing with reversed B-cell FluoroSpot. You cannot evaluate several different antigens at the same time. You need our peptide tag sequences for that. We are working on cool solution for this but it is not ready yet. Hopefully this autumn. 

/C

 

 

[tylsan]

Hej Christian!

1 hour ago, Christian@mabtech.com said:

Instead of doing a complicated enrichment of B-cells using beads I think you could start by just going up in cells/well to 400K. 

Noted! Thanks!

1 hour ago, Christian@mabtech.com said:

So maybe in parrallell you should set up another FluoroSpot plate were you prior to the assay do in fact perform the 3 days activation with R848+IL-2. 

I actually did this. I did a two day stimulation with the cells (I'll try 3 days next time) and finished developing that plate today. There were a lot more spots in the positive controls, but still sadly nothing with the antigen specific wells.

1 hour ago, Christian@mabtech.com said:

Doing a biotinylation of whole virus particles work fine. Here is kit that I think should work well:

https://www.expedeon.com/products/applications/immunoprecipitaion/lightning-link-rapid-biotin/

Thanks for the tip! I'll give it a try.

One more question, could the fluorescence enhancer cause the high background in some of the wells? Or other thoughts on the high background? I saw your YouTube video and really tried to make sure to get rid of the excess. 

Thanks for all the help!

/Tyler

[Christian@mabtech.com]

The increased membrane background could be related to the Fluorescent enhancer, but I am not certain. You say you have watched the Youtube video so the emptying of the plate is correct. 

Its strange, the background is there in some wells but not in others. Even within in the same dublicate (i.e one well is ok, the other has background). 

One thing: When setting up the FluoroSpot plate layout after you have done the coating overnight, there will be a moment when you empty the blocking and then start adding your coated antigens, stimuli or just cell culture medium. During this period, the PVDF membrane is sort of "exposed" to the high intensity air inside of the flow hood. There are still small amounts of blocking left in the wells but these can dry up quickly. If this happens, the membrane start drying up and there will be conseqences further down the line when you eventually develop your assay. The end result can look quite a lot as what I am seeing in your PDF. 

The same phenomenon described above is also true during all development stages. If it takes a long while to prepare a detection solution while at the same time the plate has been emptied, the PVDF can dry up. Some well will dry quicker than other depending on the "technique" used during emptying. There can be 20ul liquid left in one well and only 7ul in another, depending on how the plate was decanted. 

 

[tylsan]

Ok I suspected that the membrane drying could be an issue. I'll just have to try to be quicker then next time  

Thanks for all your help! Really appreciate it!

/Tyler

[tylsan]

One final thing, if I'm going to use a biontinylated antigen, would I still be able to detect both IgA and IgG antigen specific cells in the same well? (See attached image for clarification).

So I could follow the same protocol coating with capture anti IgA and IgG, and then when it comes time to detect cells, I could incubate with biotin-antigen and detection anti-IgG/IgA? I would not need to use 16 wells per sample either, but only 12 since I would not need antigen only wells if I am thinking correctly? I would be able to detect total IgA, total IgG and antigen specific IgG and IgA in the same well theoretically since the antigen specific spots would be two colored correct? Sort of how Peter did with identifying antigen specific IgG subclasses in the article you mentioned earlier.

Or would it be better to have separate wells for detecting IgG and IgA. In that case would a normal ELISPOT be better here since I would only need to detect one type of spot?

Or am I making this way too complicated?

/Tyler

 

[Christian@mabtech.com]

In principle this will work. However, we currently do not provide a streptavidin-Cy5 conjugate in our web shop. We have it in-house but it has not gone through validation as all our commercially released items. You would with this approach save time and cells, yes. 

But just as you say, you could initially evaluate this in ELISpot using one antigen and one immunoglobulin at a time. Maybe that is a smarter first step in order to find out if the reversed B-cell protocol is the right thing for your antigens. 

 

[tylsan]

Thanks for all your insight and tips Christian and Mabtech! It's really appreciated!