Dear all,
Recently I have been working with monoclonal antibody production and I usually Immunize mice for a total of 4 times with the last one being diluted in PBS 3days before fusion day.
Unfortunately this time My hybridoma cell line has come up with a problem for which I dont have enough cells to continue to fusion, for which I need more time to stabilize my cells.
What can be done? Is it okay to continue more immunizations?
Hello! When I immunized animals for hybridomas, I periodically monitored antibody titer (usually once a week) and took their blood sample for ELISA. Your particular molecule (which you using as antigen for immunization) can be too weak to cause immune response. Which scheme and schedule of immunization you had used?
Dear @Dimitry
1st is with CFA, 2nd and 3rd with Incomplete FA, last with just PBS 3 days prior to making hybridoma.
Unfortunately, even if the titre is okay. My SP2/0 cells are not ready, I mean I do not have enough cells due to the cell problem for which I need more time to scale up the cell line before I can proceed to fusion. In this case, should I continue immunization or just keep track of titre?
In such case I can recommend nothing, except planning your cell expansion upfront ;)
But in many sources (including experience of my colleagues) it stated, that you have 3-5 days to harvest B-cells after last shot. Moreover - in case your Sp2/0 wasted and you have no time or means to grow them at all, you can perform very risky maneuver - 1) Harvest spleens, 2) Put pulp through cell-strainer, 3) Purify B-cells on Ficoll or by sorter, 4) Freeze them in 90% FBS + 10% DMSO.