I am working on a C5-mAb production project in fish and most of the clones I got after sub-clonning are of IgM isotype Is there any method that could orient the mice to give IgG instead o IgM?
Dear Haitham,
What biological sample did you collect to obtain your antigen-specific B cell clones? I assume blood, but how many immunizations did you perform on the animal, and how much time after antigen administration did you collect the sample?
I am asking that because more than 80% of blood B cells are resting and/or naive IgM+/IgD+ B cells. Thus it is possible that you just have a problem of timing.
The protocol I refer to is that of Barbas, Burton et al:
immunizing mice of ~6 weeks of age with 50 µg of immunogen two times at 2-3 weeks of interval, and bleed the animal ca. 1 week post second injection.
Regards and best wishes.
https://books.google.com/books?id=uCNAay6pVAgC
HERE ARE THE THINGS YOU CAN TRY TO PUSH TO REPONSE TOWARDS IgG
1. IMMUNIZE WITH AN ADJUVANT/50 UG ANTIGEN (WELL EMULSIFIED) IN THE MUSCLE ONCE A MONTH FOR 2-3 MONTHS. I SUGGEST ALUM BUT YOU CAN TRY FREUNDS.
2..Then boost after a delay of 2 months with a lower dose of antigen and use draining nodes and spleen (NOT BLOOD) to make your hybridomas. Harvest them a 4-7 days after the final boost.
The use of adjuvant, nodes/spleen and the lower final dose should push the memory clones. This is well described many books on antibody production. Read a bit!
Hi Giacomo, I am using this immunization schedule
I use the 80 day protocol where I give the mice 4 shots at (1,21,42 and 62 days) the first is in complete adjuvant, the 2nd and 3rd are in incomplete adjuvant and the last one is in PBS.
I heard about the protocol where they collect the iliac lymph node of mice instead of spleen , do you think it differs.
The collection from lymph node instead of spleen would require a different protocol of immunization: only one immunization is needed and the lymph node can be collected ca. 15 days after antigen administration.
In contrast, about 3 immunizations are needed to isolate antigen-specific B cells from the spleen.
I don't know what cloning method you are using, and therefore the efficiency of chain amplification/number of clones. However, iliac lymph nodes have in general fewer ag-specific B cells than the spleen, so it could be best to collect the samples from more than one animal.
Maybe this publication by Sado et al. can be of help.
Article Lymphocytes from Enlarged Iliac Lymph Nodes as Fusion Partne...
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