Dear all,
I transformed with EBV B cells from total PBMC and after 1 month I isolated IgG+ cells using Miltenyi beads.
Keeping in culture these cells with CpG and without, I got proliferation but I didn t get any antibody production. I tested specific antigens (from MV and TT) that were supposed to give a response. Also total IgG levels were negative.
Anyone knows how this can be?
Thank you in advance
Alessia
I speculate the immortalized B cells transfected with EB virus no longer possess the ability to secrete IgG due to the hypermethylation of the promoter region of the genes of interst.
I supposed that you didn't inmortalized B cells specific for the antigen that you are looking for.
a few years ago I tried in LCL culture medium but I was not able to find IgG. I think that the consideration of Go J Yoshida is correct, but I also suspect that the antibody concentration in the culture medium could be very low (I used a nephelometer, which is not very sensitive).
I agree with Yoshida that immortalized B cells have no posibilites to secrete IgG
I agree completely with Ana Maria Moro. Our EBV cell lines produce differents types of Ig but they stop producing them after a time.
Does anyone has experience on isolateing single cell clone from LCL cells, I tried many methods and repeated several times, but without success.
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