positive control reference value

[Guest Eunju, Jung]

Hi, thank you for your kind answer about previous my question.

 I'm writing again to ask another question. It's first time to me conducting B cell ELISPOT, so many things are confusing me.

I wounder the positive control (PC) count reference value.

I will use the Human IgG ELISPOT basic (product code:3850-2H) to measure influenza Ag specific memory B cell and will use monoclonal antibody MT91/145 as a positive control.

I'm using cryopreserved PBMC and blood samples were drawn at prevaccination and 28 days post vaccination against influenza.

And I will plate cells at 10,000/100ul per well for positive control.

I will compare cell response at prevaccination and 28 days post vaccination, and I think PC may be different with each other.

I hope you can give an information about PC reference value. It will be a big help.

 

Sincerely,

Eunju, Jung

[Christian@mabtech.com]

Dear Eunju, welcome back!

It is difficult to provide reference values in cellular assays. We can show you some typical results that we have gotten in-house and if you look in the webshop at 3850-2A (same products as yours but ALP instead of HRP) we show you such an in-house result from Mabtech. In our example, total IgG was analyzed in ELISpot after PBMC had been incubated for 3 days together with r848 and recombinant IL-2. Cells were washed and then added at 50,000 cells/well. This resulted in around 355 spots in this donor:

https://www.mabtech.com/products/human-igg-elispot-basic-kit-alp_3850-2a

Thus, I think your plan of using 10,000 cells/well for total IgG could be a bit low. 

Since you in the B-cell ELISpot can analyze both total IgG secreting B-cells and antigen specific B-cells, you get this nice ratio of total versus antigen specific! Something you do not get in T-cell ELISpot. Its like an internal assay control. If the number of total IgG spots are zero, you cannot expect to have any antigen specific spots either.  

Are you planning on incubating your PBMC samples with r848 and IL-2 prior to running your assay? Pre-vaccination this will be required, and 28 days post vaccination there are no circulating plasmablasts in the peripheral blood anymore (they go away around day 7-10 if I am not mistaken), so pre-activation will be required here as well. 

Are you coating with the influenza antigen or will you use a reversed B-cell ELISpot with biotinylated antigen?

best,

Christian   

 

 

[Guest Eun ju, 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 as your previous comment, using R848 and IL-2 for prestimulation (for 72 hrs).

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

I have a 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.

* 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.