High IgM Background in IgA/G/M Fluorospot kit

[Leon]

Hi Christian,

We are currently experiencing high background with the IgM detection within our IgA/G/M fluorospot.

This issue seems to present wherever cells have been plated 

IgA and IgG detection works well !

We have followed the Mabtech IgA/G/M fluorospot kit protocol exactly

Any ideas on how the background (non specific binding) for IgM-cy5 can be reduced ?

Attached are our plate fluorospot read outs and plate plan

Thanks

Fluorospot issue Cy5.pptx

[Christian@mabtech.com]

Dear Leon, welcome to the Forum!

I pressume you have stimulated PBMC for 3 days in R848+recIL2, washed cells and then added them into the FluoroSpot plate? Or are these in vivo activated somehow?

What is the number of cells/well?

With IgM you often get secretion from B-cells in unstimulated cells. It is not all IgM B-cells that do this, but a it just takes a tiny fraction and you get a lot of spots in the unstimulated wells when looking at total IgM. 

Furthermore, a fraction of secreted IgM have this property of being "sticky". They bind to pretty much any surface. This is probably what has happened to you in this experiment. That is why you get spots even tough you just coated with PBS only. Some IgM stick to the PVDF membrane and stay there despite washing. They in turn get visulized by the Cy5 detection antibody. 

 

[Leon]

1) Yes, we stimulated the PBMC for 3 days with R848 (0.5ug/ml) + IL2 (5ng/ml), washed and added to plate.

2) 2x10^5 cells for PBS and antigen coated wells. 1x10^4 cells for Total IgA/G/M

I understand that IgM is 'sticky' due to its pentameric structure and binding, but we really want this to work as it will complement out optimised IgA, IgG and IgM ELISAs 

Are there any further tips you could suggest ?  

   

[Christian@mabtech.com]

Ok, got it. Since IgG and IgA look technically very good, nothing to fault. 

I do however have one good tip:

You should try something we call reversed B-cell FluoroSpot. Instead of coating with antigen, you label the antigens with "tags". We in turn have great antibodies recognizing these tags, in turn conjugated to fluorochomes. 

Or alternatively, if you are only interested in one antigen you can label it with biotin (easy to do, many bioting labeling kits on the market) and then use our SA-550 to detect spots. 

The benefit of this approach is that you often get increased sensitivity, use less antigen and the backgorund problem of IgM goes away. 

Here is open source publication made by one of our very good Phd students at Mabtech:

https://www.ncbi.nlm.nih.gov/pubmed/26930550