B cell ELISPOT, positive control

[Guest Eunju, Jung]

Thank you for your previous kind reply.

I'm working B cell ELISpot with PBMC (pre-vaccination and 28 days post vaccination against influenza), and using R848 and IL-2 for prestimulation for 72 hrs as your previous comment.

Actually, I'm using  a ELISpot kit (3850-2H), including monoclonal Ab MT91/145. 

I have an another question. In my ELISpot , positive control spots increase by 100 (60~200), comparing prevaccination and postvaccination.

I have a difficulty interpreting positive control, so I wonder markers of positive control.

Is it normal that PC spots show an increase between pre and post vaccination? (Can it be explained by an expansion of B cell after vaccination?)

* Regarding previous topic, I coated with influenza Ag instead of using a reversed ELISpot.

 

Sincerely, 

Eunju, Jung

[Christian@mabtech.com]

Dear Eunju,

Sorry about missing your previous response in the other thread. For some reason, no notification reached my email. But I got one when you started a new thred...

Anyway, an increase in the number of positive control spots post vaccination does sound reasonable in my mind. You essentially provide a "boost" when doing the vaccination and the number of circulating B-cell specific for your influenza antigen increases. It makes sense.  

Furthermore, it is our experience that you will have B-cells actively secreting IgG post vaccination for around 2 weeks. After this, they seem to leave the circulation and at 28 days they are gone. These B-cells do not require pre-stimulation with R848 and IL-2 for 3 days. You just draw blood, isolate PBMC and add them to influenza coated wells. You will see spots during the first 2 weeks. 

However, at 28 days I still think a vaccination can have induced the immune system to increase the number of memory B-cells in circulation. An increased proportion of these cells and you will observe more spots in you Total IgG ELISpot.