Hybridoma are cells that have been engineered to produce a desired
antibodyin large amounts. To produce monoclonal antibodies, B-cellsare removed from the spleen of an animal that has been challenged with the relevant antigen. These B-cells are then fused with myelomatumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer). This fusion is performed by making the cell membranes more permeable. The fused hybrid cells (called hybridomas), being cancer cells, will multiply rapidly and indefinitely and will produce large amounts of the desired antibodies. They have to be selected and subsequently cloned by limiting dilution. Supplemental media containing Interleukin-6 (such as briclone) are essential for this step.
Selection occurs via culturing the newly fused primary hybridoma cells in selective-media, specifically media containing 1x concentration HAT for roughly 10-14 days. After using HAT it is often desirable to use HT containing media. Cloning occurs after identification of positive primary hybridoma cells. Clone by limited dilution. While some may believe that IL-6 is essential for this step, it is not necessary to add that expensive supplement, rather use 50% heat-inactivated FBS for the first week. Add 10% FBS DMEM to the clone culture plate after screening for single colony wells.
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