Total mouse IgG ELISPOT - not many spots?

[Guest Lucy]

Hi there,

Thank you to Mabtech for hosting this very helpful forum!

I am interested in using ELISPOT to detect antigen-specific B cells after mouse immunization. As a first step I am trying to detect total IgG from polyclonally stimulated C57BL/6 splenocytes. However, I think I am having sensitivity issues with my assay. I do see spots, but far fewer than expected. Annotated images from our AID reader are attached.

I used MSIPS4W10 plates that were activated with 35% ethanol and coated overnight with 10 ug/ml capture anti-mouse IgG1. The splenocytes were activated in vitro with 0.5 ug/ml R848 and 5 ng/ml for 3 days and were >90% viable. I washed them once and plated varying numbers on RPMI 10% FCS blocked plates for 24h. After that I added 0.5 ug/ml biotinylated detection anti-mouse IgG1 in 0.5% FCS/PBS for 2h, followed by 1:2000 Streptavidin-HRP for 1h, then developed using KPL TrueBlue (precipitating) Peroxidase Substrate. All washes in between were with PBS no Tween.

Do the responses look typical for this kind of assay? How can I improve the sensitivity?

Thanks in advance for your advice!

Mouse IgG ELISPOT.pptx

[Christian@mabtech.com]

Hi Lucy, welcome to the forum!

No this certainly looks strange. Way too low spot count. 

Where have you purchased the polyclonal antibodies against mouse IgG1?

At only 50K cells/well, our in-house results look like this:

[Guest Lucy]

Thanks for your reply Christian!

I am using a monoclonal anti-mouse IgG1 antibody pair from BD - capture #553445 and detection #553441. We had them in the lab already and I thought in theory it should work - but I guess it's not ideal. Do you think this is why the assay didn't work well?

[Christian@mabtech.com]

Hi again Lucy,

Yes, definitly, this could be the reason. Some antibodies simply dont work well in ELISpot. When we develop new antibody systems we can have 8 different pairs that perform well in ELISA. There are differences, but not massive. However, testing the same 8 pairs in ELISpot and only 1or2 works well. 

ELISpot requires the absolute best antibodies to work well. That is why our portfolio of Mabtech monoclonals tend to be among the best in the market. 

Buy our kit and repeat!

[Guest Lucy]

Thanks for your reply Christian - my Mabtech kit just arrived!

This is probably a basic question, but if I want to detect total IgG-secreting cells, would i need to activate B cells in vivo? Or is there already a sufficient baseline level of total IgG-secreting cells in unimmunized mice for ELISPOT detection?

[Christian@mabtech.com]

Hey Lucy!

For unimmuniced mice you absolutly have to activate B-cell in vitro using our stimulation cocktail. Otherwise, there will be pretty much no spots in the ELISpot. 

Good luck!

[Guest Lucy]

Hi again, just wanted to give an update - it worked, thank you so much! I tried to attach some pictures but the editor doesn't seem to be working?

In case it's helpful for anyone, I stimulated C57BL/6 splenocytes with 1 ug/mL R848 and 10 ng/mL IL-2 (as recommended) at 2 million cells/mL for 4 days and plated the cells for 24 hours. I strictly followed the Mabtech protocol and used strep-HRP at 1:500 and developed plates with TMB for 10 minutes. 

Now for the harder task of detecting antigen-specific IgG B cells!

 

[Christian@mabtech.com]

Dear Lucy! 

This is fantastic news. Easiest to attach things is to put them into a PPT and then upload it. 

[Guest Lucy]

Hi Christian, that's what I'm trying to do, but the uploader is stuck on "Loading..." and I can't seem to attach anything?

[Christian@mabtech.com]

That is unfortunate. I checked our setting and Guest should be allowed to upload attachments, no restrictions. 

However, I did my own testing and yes, the upload function dont seem to work when posting as a guest. This worked before with earlier versions of the forum software. I will contact our supplier and ask for an update.